Design and Development of Polysaccharide-Doxorubicin-Peptide

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Design and Development of Polysaccharide-DoxorubicinPeptide Bioconjugates for Dual Synergistic Effects of Integrintargeted and Cell-penetrating Peptides for Cancer Chemotherapy. Alexandra A P Mansur, Sandhra Maria Carvalho, Zelia Ines Portela Lobato, Maria de Fátima Leite, Armando da Silva Cunha, and Herman S. Mansur Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00208 • Publication Date (Web): 23 May 2018 Downloaded from http://pubs.acs.org on May 23, 2018

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Bioconjugate Chemistry

Design and Development of Polysaccharide-Doxorubicin-Peptide Bioconjugates for Dual Synergistic Effects of Integrin-targeted and Cell-penetrating Peptides for Cancer Chemotherapy Alexandra A. P. Mansur†, Sandhra M. Carvalho†,§,#, Zélia I.P. Lobato§, Maria de Fátima Leite#, Armando da Silva Cunha Jr‡, Herman S. Mansur†,∗ †

Center of Nanoscience, Nanotechnology and Innovation - CeNano2I, Federal University of Minas GeraisUFMG, Av. Antônio Carlos, 6627 – Belo Horizonte/MG, Brazil. *e-mail: [email protected] (HSM). § Department of Preventive Veterinary Medicine, Veterinary School, Federal University of Minas Gerais, Brazil. # Department of Physiology and Biophysics, ICB, Federal University of Minas Gerais, Brazil. ‡ Department of Pharmaceutical Products, Faculty of Pharmacy, Federal University of Minas Gerais, Brazil.

Abstract Polymer-drug conjugation is an attractive approach for target delivering insoluble and highly toxic drugs to tumor sites to overcome the side-effects caused by cancer chemotherapy. In this study we designed and synthesized novel polymer-drug-peptide conjugates for improved specificity on targeting cancer cells. Chemically modified polysaccharide, carboxymethylcellulose (CMC), was conjugated with doxorubicin (DOX) anticancer drug by amide bonds and dually biofunctionalized with integrin-target receptor tripeptide (RGD) and L-arginine (R) as cell-penetrating amino acid for synergistic targeting and enhancing internalization by cancer cells. These bioconjugates were tested as prodrugs against bone, breast and brain cancer cell lines (SAOS, MCF7 and U87) and a normal cell line (HEK 293T, reference). The physicochemical characterization showed the formation of amide bonds between carboxylates (-RCOO-) from CMC biopolymer and amino groups (-NH2) from DOX and peptides (RGD or R). Moreover, these polymer-drug-peptide bioconjugates formed nanoparticulate colloidal structures and behaved as “smart” drug delivery systems (DDS) promoting remarkable reduction of the cytotoxicity towards normal cells (HEK 293T) while retaining high killing activity against cancer cells. Based on cell viability bioassays, DNA-staining and confocal laser microscopy, this effect was assigned to the association of physicochemical aspects with the difference of the endocytic pathways and the drug release rates in live cells caused by the biofunctionalization of the macromolecule-drug systems with RGD and L-arginine. In addition, chick chorioallantoic membrane (CAM) assay was performed as an in vivo xenograft model test, which endorsed the in vitro results of anticancer activities of these polymer-drug systems. Thus, prodrug nanocarriers based on CMC-DOX-peptide bioconjugates were developed for simultaneously integrintargeting and high killing efficacy against cancer cells, while preserving healthy cells with promising perspectives in cancer chemotherapy. Keywords: Polymer-Drug Conjugate; Polymer Bioconjugate; Drug Delivery Nanocarrier; Cancer Chemotherapy.

∗ To whom correspondence should be addressed: Federal University of Minas Gerais, Av. Antônio Carlos, 6627 – Escola de Engenharia, Bloco 2 – Sala 2233, 31.270-901, Belo Horizonte/MG, Brazil; Tel: +55-31-34091843; Fax: +55-31-34091843; E-mail: [email protected] (H. Mansur)

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1. Introduction Despite indisputable scientific and technological advances in recent decades, cancer remains one of the leading causes of deaths worldwide. Conventional cancer therapies such as chemotherapy has had limited success in the treatment of cancer over the years, mainly due to the focus on intensive cell killing without high specificity and often result in severe side-effects and toxicity. Therefore, there is an urgent need for the development of novel tumor-targeted therapies that could effectively and specifically target tumor cells with the lowest possible toxicity of the therapeutic agent towards normal cells.1-10 The design of tailor-made macromolecule-drug conjugates provides a synthetic approach that can overcome most of the drawbacks of drug non-selectivity in traditional chemotherapy. Several alternatives of tumor-targeting macromolecule-based anticancer drug conjugates have been developed aiming at high specificity towards cancer tumors, minimizing side-effects to healthy tissues and organs while maintaining the effectiveness of the selected drug for killing cancer cells. Among several drugs, doxorubicin (DOX) is an effective and broadly used anticancer agent for treating different tumors, which has been considered as the gold standard in oncology for chemotherapy and often adopted as a model anticancer drug for developing and evaluating macromolecule-drug conjugates. In addition, DOX has inherent optical properties due to the anthracycline chromophore group that can be used for imaging purposes providing information on drug distribution in cells and tissues.2-7,11 Since the hallmark of Ringsdorf´s paper12 proposing a polymer conjugate model, the field of polymer-based drug conjugates for drug delivery in cancer therapy has received increasing interest over the past few decades. Essentially, the model consists of a biocompatible polymer backbone, a solubilizer moiety, a covalently bound drug (with or without linker), and a targeting moiety for the construction of multifunctional targeting drug delivery systems (DDS).13 This concept has been one of the most effective and prevailing approaches to overcome the drawbacks of drug delivery systems in traditional cancer chemotherapy.14 This field of research has significantly

evolved

since

the

first

synthetic

polymer–drug

conjugate

N-(2-

hydroxypropyl)methacrylamide (HPMA)-doxorubicin conjugate entered in clinical trials in 1994, where several other polymer–drug conjugates, such as polyglutamic acid (PGA), poly(lactic-coglycolic acid) (PLGA) and poly(ethylene glycol) (PEG) have been reported in the literature.15-19 However, only few polysaccharides-based polymer-drug conjugates (e.g., hyaluronic acid, dextran and cyclodextrin) have been explored and effectively reached clinical trials in oncology medicine.15 More recently, there have been considerable interests in developing biodegradable polymers such as polysaccharides for effective drug delivery systems as they are natural compounds, 2 ACS Paragon Plus Environment

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nontoxic, inherently biocompatible and widely available. Among them, cellulose (a polysaccharide consisting of a linear chain of several thousands of β(1→4) linked D-glucose units), which is renewable, sustainable, and eco-friendly is one of most abundant natural polysaccharides. However, cellulose has limited solubility in water and therefore, restricts some of its use for pharmaceutical and biomedical applications. Thus, cellulose derivatives, such as carboxymethylcellulose (CMC) can be used as chemically modified polysaccharides for developing polymer-based therapeutic conjugates. CMC is inexpensive and broadly used in many technological fields such as, pharmaceuticals, nutrition, agriculture, etc. In addition, CMC has been proven to possess good biocompatibility and thus it is a promising material for biomedical applications. CMC is a water-soluble polysaccharide in practically the full range of pH from acidic to alkaline solutions, including at physiological conditions, possessing both carboxylate and hydroxyl groups that allow this multifunctional macromolecule to exert strong interactions with drug molecules.5,20-21 The covalent conjugation of anticancer molecules to polymers producing macromolecular-drug structures reduces its immunogenicity, improves its aqueous solubility, increases its stability and circulation time in blood and reduces the toxicity, enhancing the therapeutic value of the anticancer drug. Although, almost all the polymer–drug conjugates exploit the passive targeting, active approaches with targeting ligands such as peptides, proteins and folates have been developed in the last years. In addition, new promising lines of research are being developed to achieve active targeting by polymer–drug conjugates that inhibit specific kinases, activate apoptosis and decrease angiogenesis.7,15 Among several alternatives of targeting ligands for therapy in cancer, a large number of peptides and peptidomimetics based on the RGD (Arg-Gly-Asp) recognition sequence were developed in the past two decades as integrin ligands. Integrins are heterodimeric cell surface receptors that mediate cellcell and cell-extracellular matrix adhesion. These molecules play a key role in processes such as cell growth and proliferation, differentiation, migration, cell trafficking, besides contributing to tumor angiogenesis and metastasis. Due to their essential role in biological processes, integrins have been the subject of innumerous studies as amenable targets in medicinal chemistry aimed at the discovery of novel cancer therapeutics.4,22-24 In addition, RGD sequence binds preferentially to the αvβ3 integrin that is expressed on tumoral endothelial cells as well as on some tumor cell types such as glioblastoma, melanoma, ovarian, breast, and prostate cancer.25 Therefore, targeting integrin receptors of highly lethal malignant tumors such glioblastoma with RGD-based prodrugs can offer promising diagnostic and therapeutic perspectives for improving the current prognostic of patients.25 Moreover, over the past 20 years, cell-penetrating peptides (CPPs) have captured the attention of 3 ACS Paragon Plus Environment


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biomedical researchers, biophysicists, biochemists and chemists as these molecules, such as the class of arginine-rich CPPs, efficiently enter cells and mediate entry of macromolecules that by themselves do not cross the plasma membrane. However, despite known that endocytosis plays a key role in the cellular uptake phenomena, the mechanism involved is yet not completely understood and several key questions remain unanswered.26,27 Unexpectedly, although polymer-drug conjugates have been subject of crescent interest in recent decades, few studies were published using cellulose and derivatives as prodrug nanocarriers for cancer therapy,28-31 but no research was found in the consulted literature using carboxymethylcellulose conjugated with doxorubicin and peptide-modified for specific targeting and killing integrin-rich cancer cells. Thus, we report the rational design and synthesis of novel polysaccharide-drug bioconjugates based on CMC-DOX conjugates with RGD and L-arginine modifiers developed as drug delivery nanocarriers for specific integrin-targeting and killing cancer cells to improve the chemotherapeutic efficiency and minimize the side effects on normal cells, which offers extraordinary perspectives in cancer chemotherapy.

2. Results and discussion 2.1. Design and synthesis of polymer-drug bioconjugates In this study, novel polysaccharide-drug bioconjugates based on CMC-DOX coupled with RGD and L-arginine (R) modifiers were designed and synthesized. The aim was to combine a set of desirable physicochemical, biochemical, and chemotherapeutic properties into a single integrated macromolecule structure. Using the Ringsdorf model, carboxymetly cellulose (CMC) polysaccharide was selected as the biocompatible water soluble polymer backbone carrier with the DOX, a fluorescent standard chemotherapeutic drug, covalently bonded via chemical reactive groups. As targeting biomolecule and penetrating moiety, RGD and L-arginine, respectively, were also covalently bonded to the CMC polymer. The biopolymer was selected with lower degree of substitution (DS = 0.7) to favor biodegradability and, with a molecular mass (Mw = 250,000 Da), above the renal threshold, to prevent polymer-drug conjugates from rapid blood clearance and elimination from the body considering perspective clinical applications of these designed systems. In addition, according to the literature, the relative high molecular molar therapeutics may preferentially accumulate in tumors increasing the therapeutic action in vivo due to the enhanced permeation and retention (EPR) effects.16,32-35 Moreover, based on previous studies of our group, using fluorescent quantum dots


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directly stabilized with CMC in aqueous media,36,37 this cellulose derivative macromolecule can be internalized in tumoral and non-tumoral cells without the presence of transmembrane vectors. The carboxyl-reactive 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) was utilized for covalently coupling the amine groups from DOX, RGD and L-arginine (R) to the available carboxylic groups present in the CMC structure (R-COOH) through amide bonds, leading to the formation of the polymer-drug conjugates. Moreover, EDC is a water-soluble, “zero-length” crosslinker that is not incorporated into the conjugated structure by the reaction, and therefore, there are no concerns about its toxicity compared to other crosslinkers (glutaraldehyde, formaldehyde, epichlorohydrin, etc.).38,39 In addition, this type of linkage between polymeric carrier and drug was selected due to the possible enzymatic cleavage of amide bonds by cancer cells reported in the literature for other polymer-anticancer systems.34 The structures of the designed biomacromolecules based on CMC-DOX modified with RGD and R (CMC-DOX-RGD-R) are schematically depicted in Figure 1. The chemical formulas of precursors (CMC, DOX, RGD, R, and EDC) are presented in Figure S1 (Supporting Information) and the schematic representations of prodrug intermediates (CMC-DOX and CMC-DOX-RGD) are shown in Figure S2 (Supporting Information). Based on the literature,40 the average the amount of DOX in CMC-conjugates was 3 mg/g (DOX:CMC drug-loading content, DLC) and drug-loading efficiency (DLE) was calculated 98.0 wt. %, indicating high drug encapsulation efficiency of the polymer-drug bioconjugates.

2.2. Physicochemical characterization of bioconjugates 2.2.1. FTIR spectroscopy characterization Fourier transform infrared (FTIR) spectroscopy was performed to characterize the chemical reaction leading to the formation of the prodrug conjugates. The FTIR spectrum of CMC is presented in Figure 2a and shows a strong band centered at 1593 cm-1 associated with carboxylates (COO−) asymmetric stretching, which is expected as the CMC carboxylic acid groups (–COOH bands at 1730 cm-1) are mostly deprotonated at the pH=5.5 (pKa ~ 4.6) used for the conjugation.37,41 Upon conjugation with DOX, changes in the macromolecular structure of CMC can be observed by infrared spectroscopy (Figure 2b). After the EDC mediated reaction, two absorption bands associated with the formation of amide bonds at 1645 cm-1 (C=O, Amide I) and 1565 cm-1 (NH and CN, Amide II) appeared.42 In addition, the presence of vibration bands related to DOX at 1710 cm-1, 1617 cm-1, 1585 cm-1, and 1540 cm-1 indicated the incorporation of the anticancer drug in the macromolecular structure.43-45 These FTIR results demonstrated the formation of the amide linkage between the COO5 ACS Paragon Plus Environment


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group of CMC and amino group (NH2) of the drug. The FTIR analysis of bioconjugates of CMCDOX modified with RGD sequence (Arg-Gly-Asp) and L-arginine are less evident, because the peptides exhibit characteristic vibrations of asymmetric carboxylate and primary amine (-NH3+ antisymmetric and symmetric stretching modes and N-H bending) in the range of 1500 cm-1 up to 1640 cm-1, which are overlapped with CMC, DOX and amide bonds in FTIR spectra (Figure 2c and 2d). However, it is noteworthy the expressive increase on the intensities of the bands of amide bond in CMC-DOX-RGD-R (Figure 2d) due to the higher amount of amino acid species available for conjugation with the CMC polymer in comparison to DOX or RGD molecules.

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Figure 1 - Schematic representation of the polymer-drug conjugates based on CMC and DOX with RGD-targeting and L-arginine-penetrating moiety modifiers (CMC-DOX-RGD-R) according to Ringsdorf model. 6 ACS Paragon Plus Environment

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NH 1670

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Figure 2 – FTIR spectra of (a) CMC, (b) CMC-DOX, (c) CMC-DOX-RGD and (d) CMC-DOXRGD-R.

2.2.2. UV-Visible spectroscopy characterization Doxorubicin is a popular research tool due to its inherent absorption and fluorescence associated with the central anthracycline chromophore group. DOX is tetracyclic, containing three planar and aromatic hydroxyl anthraquinonic rings that compose its chromophore, as well as one nonplanar, nonaromatic ring attached to an aminoglycosidic side chain.46 Visible absorption spectra of DOX (a), CMC (b) and prodrug systems (CMC-DOX (c), CMC-DOXRGD (d), and CMC-DOX-RGD-R (e)) are shown in Figure 3A. The intrinsic DOX absorption characteristic resonance peaks were observed at λ=450 nm, 484 nm, 497 nm, and 534 nm47,48 in the blue-green range of the spectrum rendering, as expected by the color wheel, the orange-red color characteristic of DOX (Figure 3B). These peaks are associated with π→π* energy state transitions of quinonoid structure48,49 and were also verified in all of the prodrug systems (Figure 3A(c-e) and Figure 3C) indicating that the DOX remained stable after conjugation to the polymer. As a reference, CMC solution is optically clear and transparent (Figure 3D) without energy transitions in the visible range of the spectrum (Figure 3A(b)). 7 ACS Paragon Plus Environment


(A)

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(e)

(D) (d) (c) (b)

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Figure 3 – (A) Visible absorption spectra of (a) DOX, (b) CMC, (c) CMC-DOX, (d) CMC-DOXRGD and (e) CMC-DOX-RGD-R. Digital images of (B) DOX, (C) CMC-DOX prodrug and (D) CMC solutions.

2.2.3. Photoluminescence spectroscopy (PL) characterization Figure 4 depicts fluorescence spectra of pure DOX (a), CMC (e) and of prodrug conjugates (b-d). DOX spectra presented a maximum emission at λ = 594 nm red-shifted by 110 nm from the maximum of absorption spectra (Stokes shift), compatible with DOX in dilute solutions.50 On the contrary, CMC polymer presented a neglected fluorescence emission background due to the absence of unsaturated bonds of conjugated π-electrons as chromophores. The results also indicated the preservation of the DOX fluorescence signature after the DOX conjugation forming the CMC-DOX prodrugs but with the typical quenching of the PL intensity measured by quantum yield (QY).51,52 The calculated QY values were 10.8 ± 1.8 %, 3.5 ± 0.2 %, 3.4 ± 0.1 %, and 2.4 ± 0.1 % for DOX, CMC-DOX, CMC-DOX-RGD, and CMC-DOX-RGD-R, respectively. The QY of DOX was in agreement with literature,50 and the significant reduction of approximately 68 % for CMC-DOX and CMC-DOX-RGD systems, and ca. 78 % for CMC-DOX-RGD-R complex were detected. As the 8 ACS Paragon Plus Environment

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DOX molecule behaves as an active fluorophore, PL spectroscopy has been successfully used to study conformational alterations and interactions of DOX with its surroundings, for instance, with DNA, RNA, peptides/proteins, membranes, and drug carriers.46,53 Thus, here, we demonstrated the effective binding of DOX to CMC polymer by the formation of conjugates affecting the optical emission through fluorescence quenching due to molecular interactions.

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Figure 4 – PL emission spectra of (a) DOX, (b) CMC-DOX, (c) CMC-DOX-RGD, (d) CMC-DOXRGD-R and (e) CMC polymer.

2.2.4. Particle size and Zeta potential measurements Specially designed polymer-drug systems aim to mimicking the ability of nature to create functional and dynamic superstructures of macromolecules with tunable conformation and size by the combination of hydrophobic and hydrophilic domains and complex balance of charges.54 For that reason, the study of intermolecular interactions in solution is of central importance to most chemical and biochemical processes. The solution behavior of molecules, their stabilization or aggregation, complex formation, and/or interaction with the solvent, is a consequence of the balance of intermolecular interactions and crucial when considering their biological activity. Physicochemical characteristics such as molecular mass, particle size, morphology, zeta potential, and surface hydrophobicity are key aspects that contribute to the overall behavior of the system in vitro and in vivo. Amphiphilic macromolecules such as polymers offer a robust way to regulate the size, shape, 9 ACS Paragon Plus Environment


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and surface chemistry of the resulting nanostructures. Furthermore, by mixing oppositely charged amphiphilic molecules they can produce morphologies different from those formed by individual molecules.55 Therefore, in this study, we designed and synthesized polymer-drug bioconjugates by covalent coupling carboxymethylcellulose with doxorubicin forming amide bonds using precursors of opposite charges, i.e., CMC negatively and DOX positively charged. This strategy for developing supramolecular nanosized structures was analyzed by dynamic light scattering (DLS) and ζ-potential (zeta potential, ZP) measurements. Essentially, DLS technique (or photon correlation spectroscopy, PCS) is widely used for studying sizes and shapes of nanoparticles in liquids, where the time fluctuations in the intensity of light scattered by the particle dispersion are monitored. For dilute colloidal dispersions of nanoparticles, the decay rate of the time autocorrelation function of these intensity fluctuations is commonly used to directly determine the particle translational diffusion coefficient, which is related to the hydrodynamic radius of the particle (or hydrodynamic diameter, HD). In addition, zeta potential (or electrokinetic potential) is the potential difference between the medium and the stationary layer of fluid attached to the dispersed particle forming an interfacial electric double layer (DL), which is of fundamental important on studying aqueous colloidal systems.56 In this research, zeta potential measurements were evaluated varying the pH from 5.0 (pH after dialysis) to 7.4 (physiological pH) and the results are presented in Figure 5. At pH 5.0, R-COOgroups of CMC impart mostly the anionic character to this polysaccharide derivative and the ZP was (-39 ± 6) mV. Upon conjugation of CMC with DOX, an amphiphilic cationic molecule, the ZP value showed a moderate increase to (-33 ± 3) mV mostly due to the consumption of carboxylate groups for the formation of amide bonds combined with intermolecular interactions with protonated amino groups (R-NH3+) leading to the formation of electrostatic complexes. This relative small change was associated with the fact that only few carboxylate groups were consumed in the formation of amide bonds due to the limited amount of drug available for reaction in comparison with the number of COO- moieties in the biopolymer chain. In addition, for CMC-DOX-RGD conjugates, despite the reduction of carboxylate groups caused by the covalent conjugation, RGD peptides possess an overall slightly negative charge at pH 5.0 (isoelectric point, PI ~ 6.2-6.5), leading to intermediate values of ZP. On the other hand, further conjugation with L-arginine resulted in a significant increase of ZP measurements (-28 mV) assigned to the positive features of the amino acid (PI ~ 10.5) and further decrease of COO- groups of CMC involved in the formation of additional amide bonds. As the pH was increased (i.e., by addition of OH-), these ZP values were more negative by further deprotonation of carboxylic groups (COOH→COO-) of CMC polymers. 10 ACS Paragon Plus Environment

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Figure 5 –ZP as a function of pH and prodrug conjugate: (a) CMC-DOX; (b) CMC-DOX-RGD; and (c) CMC-DOX-RGD-R. (d) ZP value for all of the prodrug conjugates in PBS (pH 7.4, detail).

The average sizes of nanoparticulate structures were assessed by DLS based on the values of hydrodynamic radius (HD) for the polymer-drug-modifier bioconjugates. The systems composed of CMC-DOX, CMC-DOX-RGD and CMC-DOX-RGD-R immersed in phosphate saline buffer (PBS, pH 7.4) presented HD of approximately 76 ± 5 nm, 30 ± 5 nm and 89 ± 5 nm, respectively. In this buffer medium at physiological conditions (PBS, pH 7.4), the values of ZP were reduced compared to pH 5.0 and similar for all bioconjugates (-9 ± 4 mV). This trend was assigned predominately to the pH-sensitive behavior of polysaccharide derivative (CMC) present in the structure of the polymer-drug-peptide bioconjugates via the mechanisms of protonation and deprotonation of functional groups associated with the presence of negative and positive species (i.e., cations and anions, high ionic strength) of PBS solution balancing the overall charges in aqueous medium. The DLS results associated with the ZP measurements proved that CMC-DOX conjugates effectively formed surface charged macromolecular systems dispersed in aqueous medium. These findings can be interpreted based on the approach used for building these bioconjugates where CMC behaved as 11 ACS Paragon Plus Environment


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multifunctional chemical biomacromolecule due to the presence of carboxylic groups, forming negatively charged structures at mildly acidic and at physiological conditions. In addition, doxorubicin used as the anticancer drug, is mostly positively charged due to the presence of the amino groups at the sugar ring of the molecule. Therefore, we hypothesized that the covalent conjugation of CMC with DOX via amide bonds using EDC as “zero-length” linker formed polymerdrug bioconjugates, combined with the electrostatic and polar interactions of several chemical functionalities of CMC and DOX, caused the development of supramolecular structures (SS) as nano-sized entities (i.e., nanoparticles) dispersed in aqueous solution. According to the literature, polymer-based conjugates (e.g., CMC with drugs, proteins, etc.) can undergo to the formation of supramolecular structures referring to the structural organization of the carbohydrate-based polymer molecules beyond the individual molecule owing to their strong intra- and intermolecular interactions.57,58 To this end, the HD results assessed by DLS proved the formation of nanosized structures dispersed in aqueous colloidal medium. The conjugation of CMC with DOX formed nanoparticulate systems as the result of the combination of several factors including the electrostatic attraction between negative CMC polymer (i.e., carboxylates)

and positive DOX (i.e., amino

groups), amphiphilic interactions between hydrophilic groups of CMC with water solvent and hydrophobic regions with DOX planar aromatic structure. The overall contributions resulted in conformational changes to minimize the free energy of the polymer-drug-modifier conjugates in polar water medium, where a significant higher HD value was observed for CMC-DOX (~76 nm) than CMC-DOX-RGD (~30 nm). Moreover, by considering that ZP values were similar at pH = 7.4, this relatively larger size for CMC-DOX was attributed to the higher repulsion of carboxylate groups, which was balanced by water molecules increasing the volume of solvation layer. By coupling with RGD peptides (CMC-DOX-RGD), carboxylate groups from CMC were consumed forming amide bonds, reducing their repulsion (i.e., less negative groups in the macromolecule), leading to the contraction of the colloidal structure, i.e. HD values. However, the additional conjugation of CMCDOX-RGD with L-arginine (CMC-DOX-RGD-R) resulted in significant changes to the tridimensional conformation of the macromolecular system. Due to the predominant positive character of this amino acid introduced to these bioconjugates, the hydrodynamic size in water expanded to accommodate all interactions (e.g., hydrophilic, hydrophobic and electrostatic) of the new complex nanostructure produced. Thus, it is anticipated that the CMC-DOX bioconjugates formed colloidal nanoparticles in aqueous media, with CMC hydrophilic groups (e.g., COOH, OH) faced outwards interacting with water polar molecules (and, eventually, with other CMC chains) and the hydrophobic regions oriented towards the DOX molecules, which are “encapsulated” inside for 12 ACS Paragon Plus Environment

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accommodating the three planar aromatic rings. Similarly, RGD peptide and L-arginine incorporated to the CMC polymeric backbone via amide bonds should undergo preferential orientation also driven by noncovalent interactions (i.e., electrostatic, H-bonding, and van der Waals interactions). The schematic representation of the assembly of these novel bioconjugates forming “core-shell soft nanoparticles” in aqueous medium is depicted in Figure 6A. The transmission electron microscopy (TEM) images showed that CMC-DOX-peptides took a clear spherical morphology, with the estimated average radii ranging typically from 5 to 20 nm, similar for all CMC-DOX-based conjugates analyzed (Figure 6B). The relatively smaller size from TEM observations should be due to the dehydration of the nanoparticles in the TEM sample preparation process and the fact that DLS size (i.e. hydrodynamic radius) included the overall contribution of the polymer-drug conjugate combined with the water solvation layer forming the colloidal solution.

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Figure 6 – (A) The schematic representation of the assembly of these bioconjugates forming a “coreshell soft nanoparticle” in aqueous medium. (B) Typical TEM image of nanoparticulate polymerdrug conjugates.

In summary, these polymer-drug-modifier conjugates composed by CMC-DOX-RGD-R effectively formed aqueous colloidal nano-sized supramolecular structures, with physicochemical properties and morphological aspects suitable for potential applications as nanocarriers for delivery and targeting anticancer prodrugs. 13 ACS Paragon Plus Environment


2.2.5. In vitro drug release assay (acellular) The release profiles of both DOX-loaded prodrugs and “free DOX” at pH 5.5 (Figure 7A) and pH 7.4 (Figure 7B) demonstrated the modulated effect caused by CMC-DOX conjugates on the drug release. The chemical stability of the CMC-DOX systems was performed in aqueous medium for 7 days at pH = 5.5 for mimicking lysosomal vesicle and acidic microenvironment of cancer cells.34,40,59-61 For CMC-DOX and CMC-DOX-RGD conjugates, DOX release was approximately 16 % and no significant release was observed for CMC-DOX-RGD-R system (typically ≤ 2.5 %, statistical error of ± 1.2 %) in 7 days incubation period, as compared to over 50 % release for “free DOX”. In addition, at physiological pH = 7.4 (Figure 7B) DOX release was smaller than at acidic medium due to the slower hydrolysis reaction (c.a. 10 % for CMC-DOX prodrug) and also to the release of “free DOX” (< 45 %) once drug solubility increases under acidic conditions.62 These results are consistent with literature where it has been widely reported the high stability and very slow hydrolysis kinetics of amide bonds in polymer-drug conjugates at neutral and mildly acidic aqueous solutions.34,40,59-61 The cleavage of amide bonds (CMC-DOX) requires acidic medium and enzyme-mediated reactions (i.e., as catalyst) present at cell microenvironment (external and internal).60,61 For that reason, it is usually endorsed to perform in vitro bioassays with cancer cell cultures to advance on the study of the biological behavior of the polymer-drug systems. In addition, the smaller DOX release values observed for L-arginine modified conjugates (CMC-DOX-RGD-R) was associated with the relative higher content of amide bonds in comparison to the other prodrug systems under evaluation.

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2.3. Biological assays 2.3.1. Cell viability assays in vitro 2.3.1.1. Mitochondrial activity To screen for potential anticancer drugs or compounds, multiple assays that measure the effect on cancer 2D cell cultures are used in preclinical models (in vitro). The MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) assay is one of the most traditional tests to assess the activity of potential anticancer compounds, and it is also the most popular assay for examining compound interactions in vitro with live cells.19 Therefore, in this study, the results of cell viability of MTT in vitro assay towards the polymer-drug conjugates for three types of cancer cells (osteosarcoma, SAOS; glioblastoma, U-87 MG; breast cancer, MCF7) and normal cells (Reference, human embryonic, HEK 293T) are presented in Figure 8, which is based on the mitochondria activity of live cells. It was clearly observed a high lethality of free DOX at both concentrations (i.e., 1.0 µM and 10.0 µM, Figure 8A and Figure 8B, respectively) for all cell types tested. The cell viability responses were reduced to approximately 50 % for cancer cells (at DOX = 1.0 µM) and approximately 40 % for HEK 293T. Similarly, at higher concentration of free DOX (10.0 µM), the cell viability responses were further reduced to approximately 40 % for cancer cells and approximately 30 % for HEK 293T. Importantly, these MTT results evidenced that free DOX presented cytotoxic effect dependent on its concentration but more lethal to normal (i.e., HEK 293T) than to cancer cells, (i.e., SAOS, U-87 MG and MCF7). This is undesirable on cancer chemotherapy treatment because no specificity of the free DOX drug between normal and cancer was observed. Conversely, the novel polymer-drug bioconjugates designed in this research based on CMC-DOX systems showed a highly selective behavior. At lower concentration (1.0 µM, Figure 8A) of DOX drug covalently bonded to the CMC chains all cell viability results were typically 75-80 %, which strongly indicated that the formation of polymer-drug conjugates reduced the cytotoxicity as compared to free DOX. Therefore, these results confirmed that the strategy of developing prodrugs based on polymer bioconjugates for reducing the toxicity of anticancer agents was effective. This effect was assigned to the presence of covalent bonds in the CMC-DOX conjugates, which significantly affected the kinetics of cellular metabolism by reducing the intracellular release of DOX and, therefore, the cytotoxicity for all cell types. However, at higher concentration of DOX drug (10.0 µM, Figure 8B) in the CMC-DOX systems, a drastic cytotoxic effect was verified for all cancer cells but remarkably not to HEK 293T, which was the model normal cell line used as reference. Cell viability responses based on mitochondria activity were reduced to approximately 40 % for all cancer



cell types, similar to the free DOX tests, but remained typically from 60-80 % for normal cells (i.e., HEK 293T).

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Figure 8 – Cell viability response by MTT in vitro assay (HEK 293T; SAOS; U-87 MG; MCF7) after 24 h of incubation with free DOX and CMC-DOX conjugates at concentrations of (A) 1.0 µM and (B) 10.0 µM.


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Comparing both concentrations, i.e., 1.0 µM and 10.0 µM (Figure S3 – Supporting Information), the differences of cell viability responses for HEK 293T cells towards free DOX, CMC-DOX and CMCDOX-RGD nanoconjugates were statistically significant based on the analysis performed (Bonferroni Multiple Analysis Test, one way analysis of variance, α: 0.05). The same trend was observed for U87 MG cells and all of the prodrugs. However, no statistical difference was verified for cell viability results of HEK 293T cells tested with CMC-DOX-RGD-R conjugates at these concentrations (ca. 82% and 74%, for 1.0 µM and 10.0 µM , respectively), which demonstrated very effective reduction of the anticancer toxicity of DOX even at higher concentration toward normal cells by the polymer conjugation strategy. The images of HEK 293T (Figure S4 – Supporting Information) and U-87 MG (Figure S5 – Supporting Information) cells before and after contact with CMC-prodrugs (24 h, [DOX] = 10.0 µM]) showed cell confluence and morphology consistent with the viability responses. The lower toxicity of the CMC-DOX-RGD and CMC-DOX-RGD-R conjugates to normal cells resulted in more than 60 % of confluence and morphological features characteristic of grade 2 (mild cytotoxicity) according to the scoring system described in ISO 10933-5. On the other hand, the high lethality associated with DOX-loaded prodrugs observed for U-87 MG cells scored as grade 4 (ISO 10933-5), which means nearly complete damage of the cell layers. These striking positive results are very important as far as specificity of anticancer drug is concerned, where the CMC-DOX conjugates maintained their high efficiency towards cancer cells similar to free DOX, but the side effects (i.e., in vitro cytotoxicity) on normal cells were significantly reduced by polymer-drug-peptides conjugation strategy. This is one of the most difficult challenges to be overcome in oncology for developing new anticancer drugs for chemotherapy, where normal cells and tissues must be preserved from cytotoxic drugs, minimizing side effects while maintaining its effectivity on the tumor sites. Here, the novel design and synthesis of the polymer-drug conjugates considered that the formation of amide bonds between CMC and DOX required the cleavage of these bonds inside the cell for releasing the active drug compound. Once released inside the cell cytosol, DOX has to reach the nucleus and disrupting the DNA causing the cell death. From the chemical and biochemical perspectives, as the metabolism of cancer cells is much more active than normal cells combined with the more acidic pH of tumor microenvironment favored the higher kinetics for DOX release from bioconjugates. Although not yet reported in the literature for CMC-DOX polymer-drug systems, in similar amide-based conjugates, covalent bonds (e.g., hydrazone, ester, amide) between the polymer carrier and drug can be partially hydrolyzed to a certain extend when exposed to the mildly acidic and extracellular vesicles at tumor microenvironment (extracellular pH, pHe), which makes them potentially internalized by cancer cells.34,63,64 Moreover, after the polymer-drug 17 ACS Paragon Plus Environment


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conjugated nanoparticles of CMC-DOX are internalized by endocytic pathways into the more acidic intracellular endosomes and lysosomes, the amide bonds are further hydrolyzed by enzymatic mediated catalysis to activate the DOX drug with DNA targeting function, promoting its delivery into cell nuclei leading to death. Thus, this strategy of producing pH-sensitive CMC-DOX conjugates using covalent coupling with cleavable bonds allowed the anticancer drug to be gradually released from the endocytosed polymer-drug nanocarriers. To this end, here is considered that the DOX release occurred more rapidly in cancer cells because of their higher metabolic activity associated with lower pH and high degrading nature of the lysosomal enzymes.34,63 For that reason, the opposite behavior was accounted for the relative lower cytotoxicity in vitro for HEK 293T. Therefore, this mechanism related to dual pH-sensitive polymer-drug conjugates, (i.e., CMC carboxylate groups and CMC-DOX amide bonds) can respond to the cancer extracellular and intracellular pH gradients to simultaneously enhance cellular uptake and promote acidic triggered intracellular release of drugs, such as DOX, showing boosted cytotoxicity more specific to cancer cells.60,65 Although functional nanomaterials have been engineered to recognize cancer-specific receptors at the cellular level for diagnostic and therapeutic purposes, the significant reduction of cytotoxicity towards normal cells for HEK 293T verified in our study, as a result of the combination of these stimuli-responsive features in drug nanocarriers, was not previously reported for polymer-drug conjugates in the literature. Moreover, another important strategy developed in this study was targeting the αvβ3 integrin receptors of cancer cell membranes by synthesizing CMC-DOX prodrug conjugates covalently bonded via a “zero length” linker (EDC) with RGD tripeptides (CMC-DOX-RGD), which are integrin vectors,34 combined with amino acid arginine (CMC-DOX-RGD-R) for enhancing cell penetration.25,66 The MTT results evidenced the targeting effect caused by RGD moieties grafted to the CMC-DOX conjugates (at [DOX] = 1.0 µM) with the higher cell viability response for breast cancer cells (MCF7, low integrin) compared to higher cytotoxicity for glioblastoma cell line (U-87 MG, high integrin). These results validated by statistical analysis proved the specificity that, in addition to the “protection” effect for non-cancerous cells promoted by the presence of covalent amide bonds in the CMC-DOX conjugates, there was an additional effect on cancer cell killing by targeting in vitro using RGD, which demonstrated integrin vector affinity towards cancer cells. At higher concentrations of anticancer drug, i.e., [DOX] = 10.0 µM, this effect was not observed probably due to the lethality caused by the high dose of DOX for all cancer cell types. However, the “protection” effect towards normal cell line (i.e., HEK 293T) was evidenced mostly probably caused by the combination of physicochemical and biochemical features of RGD and R moieties with the additional amide bonds introduced to the CMC-DOX-RGD and CMC-DOX-RGD-R bioconjugates, 18 ACS Paragon Plus Environment

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which delayed the kinetics of intracellular release of DOX combined with the lower concentration of integrin membrane receptors of HEK 293T cells. Moreover, the dose-response curves (Figure 9) and IC50 analysis (Figure 10) proved the remarkable effect of the conjugation of DOX with the polysaccharide polymer chain on the cell viability response of HEK 293T used as reference normal cell type compared to all other cancer cells.

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The IC50 values of cancer cells for all of the prodrugs were higher than for the respective free DOX indicating from 4- to 7-fold greater resistance for bioconjugates (Figure 10), pointing out the modulation of drug release by conjugation strategy. In addition, an important response was observed for normal cells (HEK 293T) regarding to their resistance increased by 7-fold for CMC-DOX compared to free DOX. Moreover, distinct from cancer cell results, an outstanding resistance of HEK 293T cells was verified of 33-fold for CMC-DOX-RGD and 53-fold for CMC-DOX-RGD-R greater than for free DOX. Therefore, based on these in vitro cell viability results it can be envisioned that targeting integrin receptors of highly lethal malignant tumors such glioblastoma with RGD-R prodrugs composed by CMC-DOX-based bioconjugates can offer promising therapeutic perspectives for improving the current prognostic of patients while keeping low side-effects on normal cells, tissue and organs.25,66

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2.3.1.2. DNA Staining (Crystal Violet Staining) One cell viability assay may not be always sufficient for a precise evaluation of in vitro performance of drug compounds against cancer cells, as complex dynamic mechanisms are involved. Therefore, in addition to MTT test, crystal violet staining (CVS) cell cytotoxicity assay was performed because it has high affinity to ribose type molecules that can bind to the external surface of the DNA double helix localized in the nuclei of cells.67 To avoid redundancy with MTT assays, in the present study, CVS assay was performed with HEK 293T cells (normal cell culture) and U-87 MG cancer cells, with two concentrations of free DOX and polymer-drug conjugates (1.0 µM and 10.0 µM) and two incubation times (6 h and 24 h), and results are summarized in Figure 11. The CVS results validated the MTT findings discussed in previous sections when compared at 24 h of incubation time. For 6 h incubation time, at the lower concentration of DOX and prodrugs (1.0 µM, Figure 11A) slight reduction of cell viability (~10 %) was observed for HEK 293T cell type and larger for U-87 MG cancer cells (~20 %). At 24 h of incubation (1.0 µM, Figure 11B), free DOX showed higher toxicity of c.a. 30% for HEK 293T and U-87 MG cells, but for the polymer-drug conjugates the cell viability remained fairly unaltered compared to 6 h of incubation. At the higher concentration of 10.0 µM of anticancer compounds the cytotoxicity was higher for both normal and cancer cells, but more pronounced for free DOX compared to conjugates and also superior for 24 h (Figure 11D) of incubation time than for 6 h (Figure 11C). Interestingly, the HEK 293T cells showed increasing cell viability responses (~from 70 % to ~80 %) at lower time of incubation of 6 h, for all systems evaluated, which is assigned to the higher content of amide bonds in the conjugates. Moreover, a remarkable resistance against DOX cytotoxicity was verified for HEK 293T cells at longer period of incubation of 24 h, with polymerdrug conjugates functionalized with peptides motifs (i.e., RGD-R). These results endorsed the MTT assays of previous section evidencing that the CMC-DOX polymer-drug conjugates biochemically functionalized with both RGD and R (RGD-R) demonstrated a unique protective behavior specific for HEK 293T cells against DOX anticancer agent even at higher concentrations. Notably, these same CMC-DOX-based bioconjugates retained effective killing activity against U-87 MG cancer cells. It was observed a good correlation between MTT and CVS results, where the major trends of cell viability responses of MTT at 24 h were similar to CVS at 6 h incubation. This is suggested as a consequence of the more rapid response of CVS interaction with DNA than the effect on the mitochondrial activity. To this end, these results are more important than just presenting a great correlation with the MTT assay based on the mitochondrial activity, but because they rely on the



affinity between the crystal violet dye and the cellular DNA, which is assigned to the main cell death mechanism associated with DOX as anticancer drug.

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2.3.2. Cellular uptake of polymer-drug bioconjugates - Confocal laser scanning microscopy (CLSM) 2.3.2.1. Cellular uptake of CMC-DOX bioconjugates - Internalization and kinetics analyses Motivated by the relevant observations from previous MTT and CVS in vitro assays and to conduct a more in-depth analysis of the effect of the polymer-drug conjugates as chemotherapeutics for cancer cells, further characterization of the cellular uptake was performed using confocal laser microscopy. Doxorubicin was used the anticancer drug and also the fluorescent biomarker (excitation λexc = 568, emission λem = LP 585 nm). It is broadly known that endocytosis is the most common mechanism to all cells in the body for internalizing macromolecules (or nano-sized particles) and retaining them in transport vesicles, which later traffic along the endolysosomal scaffold. Nevertheless, there is an array of vesicular internalization mechanisms and understanding the key players to each pathway has challenged researchers for decades to bioengineer macromolecular systems for effectively drug specialized delivery.26,27 The use of inhibitors for blocking potential pathways for internalization of polymer-drugs could be an elegant strategy, which would be helpful to elucidate more precisely the complex internalization mechanisms involved.68 Therefore, a comprehensive investigation of the cellular internalization pathways and mechanisms of the polymer-drug conjugates made of CMC, DOX and amino acid modifiers (i.e., RGD and R) is beyond the scope of the current study. To this end, the choice of cells for performing the analysis was based on the in vitro MTT and CVS results considering the unique resistance showed by normal cell HEK 293T toward the CMC-DOX bioconjugates compared with U-87 MG cancer cells due to their high content of integrin receptors at biomembranes. In addition, from the biomedical perspective, this choice is justified due to the high lethality of patients with glioma cancers. Thus, the major aspect addressed was the evaluation of the different rate of DOX release affected by the formation of DDS bioconjugates, causing the verified “protection” of normal cells in MTT and CVS in vitro assays to the cytotoxic effect of the anticancer drug. To verify intracellular uptake, free DOX and all of the polymer-based prodrug complexes were investigated by confocal fluorescence microscopy for HEK 293T and U-87 MG cell lines after incubation times: 0 min, 15 min, 30 min, 60 min, 2 h, and 6 h (Figures 12 and 13, for fluorescence images, and Figure S6 and S7 – Supporting Information, for Fluorescence + Bright Field).



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Figure 12 – CLSM images of cellular uptake of free DOX (A) and prodrug systems ((B) CMC-DOX, (C) CMC-DOX-RGD and (D) CMC-DOX-RGD-R) for HEK 293T cells after incubation for (a) 15 min, (b) 30 min, (c) 60 min, (d) 2 h and (e) 6 h (scale bar = 10 µm).


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Figure 13 – CLSM images of cellular uptake of free DOX (A) and prodrug systems ((B) CMC-DOX, (C) CMC-DOX-RGD and (D) CMC-DOX-RGD-R) for U-87 MG cells after incubation for (a) 15 min, (b) 30 min, (c) 60 min, (d) 2 h and (e) 6 h (scale bar = 10 µm).



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To analyze the complex cellular uptake mechanism of the prodrug bioconjugates, we considered a simplified process composed of three major steps as schematically depicted in Scheme 1: (1) extracellular transport of drug and internalization through the cell biomembrane; (2) intracellular traffic by endosome-lysosome vesicle; (3) transport to nucleus and drug-DNA intercalation triggering apoptosis. Prodrug conjugate

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Scheme 1 – Illustration of three major stages of the complex delivery process of drug nanocarriers to cells. 26 ACS Paragon Plus Environment

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The first stage, at the initial 15 min of cellular uptake, it was predominantly related to the transport of the DOX and bioconjugates from the extracellular microenvironment (ECM) and across the cell membrane. It is expected to be moderately slower for normal HEK 293T cells than for U-87 MG cancer cells due to common assumptions of higher metabolism of tumors and, therefore, more rapid transport of nutrients into the cell.61 However, this internalization progress is much more complex, as the permeability of cell type associated with the physicochemical aspects including surface charge, hydrodynamic radius, chemical functional groups of the nanocarriers composed by CMC-DOX supramolecular structures and free DOX can be predominant and govern the initial uptake process. As expected, no fluorescence was detected before incubation (t = 0 min) related to the cell autofluorescence (Figure S8 – Supporting Information). As presented in Figure 12A(a) and 13A(a), for the HEK 293T and U-87 MG cells, respectively, incubated with free DOX, the red fluorescence associated with DOX emission was already observed at cell nucleus after 15 min, and with qualitatively increasing intensities as the time of incubation evolved (Figures 12A(b-e) e 13A(b-e)). In addition, no significant fluorescence was detected in the cytosol for the entire process. Thus, for free DOX samples, from the initial stage (stage 1, 15 min) up to 6 h, fluorescence was always mostly concentrated in the nucleus and increasing with time, indicating a “single-step” process from cell internalization to DNA binding for both cell types. These results are consistent with the literature because DOX usually undergoes diffusion across the cell membrane and, due to its high affinity for DNA, accumulates in the nucleus, which contains most of the DNA inside the cell.69 Moreover, these PL images (Figures 12A(a) and 13A(a)) were quantitatively evaluated for PL intensity (Mean Fluorescence Intensity – MFI) at 15 min calculated by image processing software (public domain, ImageJ, v.1.5+) and the results are presented in Figure 14. It was observed slight variations of PL intensities but with statistically similar values for U-87 MG and HEK 293T cells incubated with free DOX, indicating no relevant uptake difference for the normal or cancer cell types. Therefore, these results endorsed the cell viability assays (e.g., MTT and CVS) where free DOX caused high cell killing but without detectable specificity towards normal or cancer cells. Analogously, no clear difference was observed for PL responses at 15 min for all bioconjugates comparing the two cell types. Importantly, the qualitative analysis of the set of PL images at 15 min incubation time (Figures 12a(A-D) and Figure 13a(A-D)) associated with the first stage of the drug delivery process indicated a systematic lower PL intensity for free DOX than for all prodrug bioconjugates inside the cells (cytoplasm + nucleus), independent of cell type (i.e., HEK 293T and U87 MG cells). This feature was also quantitatively evaluated by the average PL intensity plot comparing these systems (Figure 14). Thus, these results showed that the conjugation of DOX with 27 ACS Paragon Plus Environment


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CMC polymer favored initial cellular uptake probably enhancing the transport of these nanocarriers across the cell biomembrane compared to free DOX. These findings can be explained based on previous studies reporting that free DOX is known to be taken up by cells through a combined process of passive diffusion and active transport mechanisms.70 The uncharged form of the drug (DOX is a weak base with pKa ~ 8.3) is mostly transported via passive diffusion across the cell membrane, where the process depends on the nature of the interactions occurring at the biointerfaces of drug-cell membrane.71 On the other hand, in order to design and building tunable drug delivery systems (DDS) the conjugation of macromolecules and particulates is used for affecting their mechanisms of cellular internalization. Basically, the cell membrane is naturally impermeable to large molecular complexes (i.e. greater than ~ 1 kDa), but cells possess a variety of active internalization mechanisms to accommodate cellular entry of such large molecular systems. Normally, the cell membrane promotes invagination to engulf molecules and extracellular fluid in an intracellular membrane-bound vesicle (or endosome) that will subsequently traffic through the cell, a process known as endocytosis.71 Therefore, here, the bioconjugates made of CMC-DOX and peptide modifiers were mostly internalized by cells through endocytic mechanism, which was accounted for the higher uptake as compared to free DOX (i.e., mostly passive diffusion). However, distinct endocytosis pathways and direct translocation through the membrane can be triggered (sometimes simultaneously and/or sequential events) depending on the overall contributions of several parameters such as surface charge, hydrodynamic radius, polymer chemical composition, functional groups, type of cell line targeted, among other properties and experimental conditions. Another aspect considered was the surface charge of the DDS system, because free DOX is an amphiphilic and positively charged molecule and CMC prodrugs showed negatively charged conjugates (e.g., ZP values ~ -10 mV in PBS, pH = 7.4), which certainly promoted different interactions at the cell membrane interface with the phospholipids and protein components.27,71 Moreover, according to the literature, receptor mediated endocytosis (RME) allows for a more rapid means of ligand targeted internalization compared to that of untargeted systems. Depending on the receptor-dependent or independent endocytic paths, the intracellular trafficking path can also be controlled. For instance, previous studies have reported specific affinity of polysaccharides to cancer cell surface receptors (e.g., hyaluronic and CD44 receptors).60,72,73 Similarly, in our study it is suggested that cancer cell surface receptors possibly may have affected and boosted the internalization of the CMC-based bioconjugates due to higher affinity with the carboxymethyl cellulose polymer (i.e., a polysaccharide consisting of a linear chain of several thousands of β(1→4) linked D-glucose units) compared to free DOX, as several glucose receptors are overexpressed by cancer cells. For that reason, although 28 ACS Paragon Plus Environment

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sustained by these reliable results, as the cellular uptake phenomena are very complex and still under intense research and debated, it is not rational to affirm indisputably the precise mechanisms that governed the rate of transport of free DOX and CMC-based prodrugs across the cytoplasmic membranes, but highlight the key aspects involved. Therefore, it was proved that, despite the lower cytotoxicity of CMC-based bioconjugates verified in MTT and CVS assays compared to free DOX, they presented higher cellular uptake, and validated the hypothesized mechanism of “cell protection” by modulating the rate of release of DOX inside the cell compartment, reducing the cytotoxicity to normal cells.

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15 10 5 0 HEK 293T

U-87 MG

Cell line Figure 14 – Mean Fluorescence Intensity values of HEK 293T and U-87 MG cells after 15 min (stage 1) of incubation with free DOX and prodrug bioconjugates.

Hence, at the second stage (stage 2), after entering into the cell upon endocytosis, the polymer-drug bioconjugates are surrounded within the early endocytic vesicles and are not directly carried into the cytosol. In this study, the second stage of the cellular process was considered from 30 min – 120 min, which was assigned to the inner accumulation of DOX caused by degradation of prodrug conjugates at cellular vesicles, release to the cytosol, and reaching the nucleus. These results are presented in Figure 12(b-d) and Figure 13(b-d). Qualitatively, it was clearly observed a gradual increase of the 29 ACS Paragon Plus Environment


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intensity of fluorescence as the incubation time evolved for all systems (i.e., free DOX and conjugates). However, it was observed that for CMC-DOX (Figure 12B(b)) and CMC-DOX-RGD (Figure 12C(b)) systems in HEK 293T cells, after 30 min, most of the fluorescence was localized at cytosol, with minor intensity at nucleus. After 1 h - 2 h (Figures 12B(c-d) and 12C(c-d)), the red fluorescence related to DOX was mostly at nucleus with only minor fluorescence intensity remaining at cytosol. This is supporting evidence that DOX was released from the prodrug systems and, upon reaching the cytosol, was routed to the nucleus. Interestingly, a very distinct feature was observed for CMC-DOX-RGD-R bioconjugates (Figure 12D), because the fluorescence indicated accumulation of the drug at cytosol independent of the time of incubation with low fluorescence intensity reaching the nucleus of the cells. These findings revealed that DOX was released in a much slower rate and also suggesting that, part of the prodrug system (CMC-DOX-RGD-R) may have been exocytosed before the amide bond cleavage between CMC and DOX. This result was consistent with the cell viability MTT and CVS assays in which CMC-DOX-RGD-R prodrug system presented the lowest cytotoxicity, explained by fact that the anticancer drug had the overall slowest release rate and therefore limited concentration of DOX reaching the HEK 293T cells nuclei. Although not directly comparable with HEK 293T cells, the distribution patterns of DOX release from prodrugs in U-87 MG cancer cells were qualitatively similar for all of the systems (Figures 13B, 13C, and 13D) evaluated. From 30 min up to 2 h, U-87 MG cells showed red fluorescence in cytoplasm and nuclei, with higher intensity in cytosol for the first 30 min and, after that, mostly localized in the nucleus. Considering the third stage of cellular uptake process, from 2 h to 6 h after incubation, a strong red fluorescence was concentrated in the nuclei of the HEK 293T cells incubated with DOX, CMC-DOX and CMC-DOX-RGD (Figure 12e(A-C)). In Figure 15 it is depicted the DOX fluorescence distribution pattern inside the cell after 6 h of incubation, where free DOX and all polymer-DOX bioconjugates presented fluorescent localized in the nuclei, except for CMC-DOX-RGD-R systems. Analogously, from 2h to 6 h, almost all the drug uptake has been released from the polymer bioconjugates and reached the nucleus of U-87 MG (Figure 13e) cancer cells. Therefore, these results demonstrated that, despite all the well-known morphological and physiological differences between tumor cells and normal cells, the cellular internalization, the intracellular release in the cytosol and trafficking to the nucleus of DOX and polymer-drug bioconjugates presented similar tendency. That means, after 6 h of incubation with all bioconjugates except CMC-DOX-RGD-R, DOX accumulated in the nuclei of tested cells. For CMC-DOX-RGD-R conjugates the behavior was even more prominent in HEK 293T cells with low red emission in the


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nucleus after 6 h incubation, which was assigned to the previously discussed “protection effect” observed in MTT and CVS in vitro assays.

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Figure 15 - Bright field (BF) and fluorescence images merged (left column) and fluorescence intensity profiles along X-X lines (right column) for HEK 293T cell incubated with (A) DOX, (B) CMC-DOX, (C) CMC-DOX-RGD and (D) CMC-DOX-RGD-R for 6 h (scale bar = 10 µm). 31 ACS Paragon Plus Environment


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At this point we highlight that in this study DOX was used simultaneously as the anticancer drug and fluorescent biomarker for bioimaging the endocytic and cell killing paths. Regarding to the entire process, it should be considered that DOX, belonging to the class of anthracyclines, has mainly three steps of action after being taken up by the cells. First, it binds to nucleic acids, intercalating the planar anthracycline nucleus with the DNA double helix; second, it interacts with topoisomerase II, an enzyme involved in DNA replication; third, it binds to the membrane, affecting a variety of cellular functions.74 Therefore, the optical properties of DOX can be more or less affected by the contributions from biological and chemical microenvironments during the entire cellular process, ranging from endocytosis to cell apoptosis after reaching the nucleus.61 To this end, we attempted to explore the kinetics profile of internalization of free DOX and prodrug systems in HEK 293T and glioblastoma cells (U-87 MG) using confocal fluorescent images (Figures 12 and 13) based on “Mean Fluorescence Intensity, (MFI)” (ImageJ, v.1.5+). The red emission of nucleus was evaluated for normal and glioblastoma cells and the results are presented in Figure 16A and Figure 16B, respectively. These profiles endorsed our previous findings indicating a low concentration of DOX localized at the nucleus at the initial period of incubation (i.e., 15-30 min), followed by a gradual increase during the next 30-60 min interval for U-87 MG cancer cells and from 30-120 min for HEK 293T cells. That indicated a relative slower accumulation rate (or lower slope of the curve) of DOX at nucleus for HEK 293T than for U-87 MG cells. This trend was predominant for most systems, but not for CMC-DOX-RGD-R where a radically distinct behavior was verified. Both cell types showed a reverse tendency with an important decrease of the DOX release from CMCDOX-RGD-R conjugates at 30-60 min interval, followed by a gradual increase in the sequence. Such reduction of DOX emission at the nucleus was attributed to the successful strategy utilized in this research of conjugating the anticancer drug (DOX) with a biocompatible polymer (CMC) for controlling and affecting the intracellular drug release. As discussed in previous section, the slower rate was primary associated with the presence of amide bonds, which needed to be broken before releasing the drug inside the cytosol and reaching the nucleus. Moreover, the conjugation of integrintarget peptides (RGD) and arginine (amino acid, R) forming the multifunctional prodrug system resulted in different endocytosis pathways from the other systems due to the additional interactions with membrane cell receptors.75-79


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In the sequence, from 1 h to 6 h, although still increasing, all release profiles presented a reduction on the accumulation rate of DOX (i.e., smaller gradient) at the nucleus reaching a plateau of “saturation” for some conjugates (“level off”) after 2 h of incubation. These profiles must be considered as the results of the entire cellular uptake process occurring with the DOX and CMC bioconjugates. A dynamic sequence of interconnected mechanisms occurred serially and simultaneously as the incubation time evolved, from the initial “pure” endocytosis (or passive diffusion for free DOX) at the very beginning to the combination of transport across the membrane, endo-lysosomal trafficking, lysosomal degradation of bioconjugates, DOX release at cytosol, DOX targeting and reaching the nucleus, and concluding with apoptosis and cell death. This last event can eventually cause quenching of the DOX emission.61 Therefore, the analysis of drug release for U-87 MG cells, assuming the total accumulation of DOX after 6 h incubation (i.e., 100% red emission), showed approximately 100 %, 75 % and 50 % accumulation at nucleus for free DOX sample, CMC-DOX and CMC-DOX-RGD/CMC-DOX-RGD-R conjugates, respectively. Analogously, the profile for HEK 293T cells, besides the slower release rate compared to U-87 MG cells, assuming the total accumulation of DOX in glioblastoma cells after 6 h incubation (i.e., 100 % red emission), showed approximately 90-100 % accumulation at nucleus for free DOX sample, CMC-DOX and CMCDOX-RGD conjugates (equivalent within the statistical variation). However, the lowest accumulation value of approximately 30 % was observed for CMC-DOX-RGD-R, which endorsed the in vitro MTT assay and validated the hypothesis that this bioconjugate followed different endocytic pathway and intracellular trafficking. This effect reduced the kinetics of DOX release and therefore showed the lowest cytotoxicity for normal cells. As reported in previous studies, besides promoting cell adhesion behavior, membrane receptors (e.g., integrins, cadherins, selectins) deeply impact a variety of signaling cellular events including those involved in endocytosis, mitogenesis, survival, and differentiation. Moreover, some receptors (e.g., CD44) have shown specific affinity towards polysaccharides (e.g., hyaluronic acid).80 Hence, in our study, it was evidenced that the novel strategy adopted of combining several characteristics from polysaccharides (CMC, a polysaccharide of glucose units), high-affinity αvβ3 integrin ligand (Arg-Gly-Asp, RGD), and cell penetrating amino acid (R) conjugated in one hybrid polymer-drug macromolecular structure provoked the unique “protection effect” for normal cells. This behavior was verified by HEK 293T cells against cytotoxic DOX anticancer drug while still retaining the cell killing efficacy against brain tumor cells. To the best of our knowledge, no similar bioconjugate systems composed by CMC, DOX and peptides (RGD and/or R) have been investigated as anticancer prodrug and presented this outstanding behavior towards doxorubicin (i.e., model anticancer drug). This exceptional behavior was 34 ACS Paragon Plus Environment

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interpreted as the consequence of the combination of several aspects incorporated to the bioconjugates including the chemical functional groups, bio-affinity moieties, surface charge and the kinetics of intracellular cleavage of amide bonds. According to the literature, other polymer-based prodrugs presented modulated release depending on the membrane interaction and components involved in cell vesicle formation. Endosomes will mature into acidic vesicles which may or may not fuse with lysosomes, which can metabolize macromolecules using hydrolytic and enzymatic reactions.60,73 The intracellular degradation of amide bonds such as in polypeptides (or proteolysis) can be achieved in lysosomes as they contain a large number of chemical species and enzymes such as proteases (e.g., cathepsins) that catalyze the biochemical process at the subcellular compartment.34,60 To this end, for the cellular uptake process, these hybrid bioconjugates with amide bonds (CMC-DOX, CMC-RGD and CMC-RGD-R) required interactions with the membrane receptors and endocytosis via endo-lysosomal pathway at acidic pH and in the presence of degradation enzymes to causing DOX release into cell cytosol. The results proved that these factors affected the different routes for free DOX and CMC-DOX conjugates. Moreover, they simultaneously favored the cellular uptake of the CMC-DOX-RGD-R and promoted specific modulation of the kinetics of intracellular release of DOX, rendering less toxic bioconjugate prodrugs towards normal cells. At this point, it is highlighted that these in vitro results were based on the design of polymer-drugpeptide systems using CMC-DOX-RGD conjugates (for targeting integrin domains at cancer cell membranes) modified with L-arginine amino acid (R) for enhancing cellular uptake, which share many of the essential characteristics of the arginine-rich cell-penetrating peptides but may present distinct cellular internalization mechanisms.26,78,79,81 Moreover, to foresee clinical application, it is important to consider the relative low stability of linear peptides such as RGD and others regarding the enzymatic degradation due to proteases present in blood, which has been significantly improved by cyclization, incorporation of stereoisomers (i.e., d-amino acid residues), and PEGylation process. 79,82

Therefore, reliable in vitro studies must be performed before in vivo assays but they cannot be

straightforwardly extrapolated for clinical use without consistent prior validation process with animal models and pre-clinical trials.

2.3.2.2. Mathematical models of polymeric-drug release for live cells in vitro Mathematical modeling of drug release can assist on understanding the effect of several parameters, such as morphology, size, surface charge, chemical composition and structure of the drug delivery system, on the kinetics of drug release83-85 In this study, free DOX and CMC-DOX polymer-drug 35 ACS Paragon Plus Environment


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conjugates were analyzed using the most commonly used models based on relatively simple mathematical approaches and that have the advantage of employing a small number of parameters. Nonetheless it can offer supporting insights about the mechanisms occurring discussed in previous sections. The results of the in vitro drug release study using live cells were fitted with Higuchi model ([cumulative drug release] vs. t

1/2

) and the Korsmeyer–Peppas model (log [cumulative drug

released]) vs. log t) (Figures S9 to S12 – Supporting Information). The obtained fitting parameters such as “KH”, “n” and “R2” for these models are summarized in Table 1. The kinetic model that best fitted the release profile data was evaluated by comparing the linear regression coefficient (R2) values of these models (Table 1). The release profiles from free DOX, CMC-DOX, and CMC-DOX-RGD bioconjugates for HEK 293T cells in vitro have shown the best fit with Higuchi's equation, with the R2 values of 0.98, 0.93 and 0.94, respectively. According to the literature, this suggests that the experimental release profiles of DOX from these systems were most probably diffusion controlled. In addition, although not the best fitted R2 values, based on the Korsmeyer–Peppas model, these samples presented values of parameter n < 0.5 indicating Fickian diffusion, whereas n > 0.5 would represent anomalous diffusion.84-86 However, CMC-DOX-RGD-R bioconjugates release profile data did not fit well with both kinetic models, which validated the results and discussion presented in previous sections, where this system exhibited a high “protection effect” against DOX drug in HEK 2093T cells.

On the other hand, the release profile data from free DOX and all CMC-DOX

bioconjugates for U-87 MG cells (i.e., glioma cancer cells) did not fit to both models tested (i.e., Higuchi and the Korsmeyer–Peppas models), which indicated abnormal diffusion transport. This behavior was assigned to the familiar concept of complex cellular metabolism associated with cancer cells, which do not match to one single predictable transport mechanism, but diverse pathways usually involved.61,84,85


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Table 1 – Release kinetics parameters using Higuchi and Korsemeyer-Peppas mathematical models of free DOX and bioconjugates after 6 h incubation with HEK 293T and U-87 MG cells.

Drug/Prodrug conjugate DOX CMC-DOX CMC-DOX-RGD CMC-DOX-RGD-R

HEK 293T KorsemeyerHiguchi Peppas KH (h-1/2) 22.1 26.4 26.3 7.4

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0.49 0.41 0.40 0.14

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U-87 MG KorsemeyerHiguchi Peppas KH (h-1/2) 36.8 19.3 19.1 16.2

R2

n

R2

0.80 0.42 0.73 0.82

0.69 0.17 0.44 0.48

0.73 0 0.74 0.58

2.3.2.3. Cellular uptake of CMC-DOX conjugates - DNA-staining with TO-PRO®-3 An additional complementary evidence of the activity of DOX released from the conjugates at cellular nuclei was based on TO-PRO®-3 staining procedure which has a very strong binding affinity for double strand DNA.87 The results of the DNA-staining based on typical fluorescence microscopy images for U-87 MG cells after 30 min incubation with the conjugates and free DOX are presented in Figure 17 (TO-PRO®-3 emission in blue color, 1st column (a); DOX red emission, 2nd column (b); 3rd column (c), the overlapping of both biomarkers). It was observed that for DOX system, after 30 min incubation, the blue fluorescence specific for staining nucleus was significantly overlapped with the red fluorescence, demonstrating the prevalence of localization of the DOX at the DNA-rich area of the cells. These results validated the major mechanism of cell killing of DOX as anticancer drug by binding to cellular DNA causing apoptosis as broadly reported in the literature.61 Regarding to the bioconjugates (CMC-DOX, CMC-DOX-RGD and CMC-DOX-RGD-R conjugates), after 30 min of incubation, relatively lower intensities were observed in the nucleus and also DOX is scattered distributed at the cytosol, which suggested distinct cellular endocytic pathways. These results endorsed the findings discussed in the previous sections, where the chemical functional groups of the CMC (e.g., -OH, -COOH), combined with the affinity peptide modifiers for CMC-DOX-RGD and CMC-DOX-RGD-R, have significantly affected the biochemical interactions at the cell membrane biointerfaces, the cellular internalization and the drug release kinetics profile.



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Figure 17 – Confocal images showing intracellular uptake and distribution of DOX (A) and polymerdrug-peptide conjugates (B, CMC-DOX-RGD) by U-87 MG cells. (a) DNA staining with TO-PRO®3 in blue color; (b) DOX red emission; (c) Merged image of both biomarkers (scale bar = 10 µm).

2.3.2.4. Cellular uptake of CMC-DOX bioconjugates - Lysosomal biomarkers As macromolecules such as polymers and conjugated derivatives are transported along the endosomal-lysosomal pathway, lysosomal membrane markers such as LysoTracker® dye (green emission, excitation λexc = 488 nm, emission λem = 505-550 nm) was used as bioimaging tool for tracking lysosome behavior associated with the endocytic mechanism of the cells. Therefore, the doxorubicin conjugates composed of CMC-DOX-RGD-R showed good co-localization with the late endosomes and lysosomes of cells, as evidenced by the presence of yellow dots in the cells caused by overlapping the green fluorescence from lysosome marker and the red fluorescence from doxorubicin (Figure 18). Considering incubation times of 30 min and 2 h, there was a notable distinction between HEK 293T and U-87 MG cells, as the bioimages showed a much higher density of endo-lysosomal vesicles (ELV) for tumor cells compared to normal cells. This behavior was accounted for the successful strategy adopted in our designed CMC-DOX bioconjugates using RGD (i.e., peptide motif Arg-GlyAsp) proving to be specific for targeting the αvβ3 integrin receptors of cancer cells. Glioma cancer 38 ACS Paragon Plus Environment

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cells overexpress integrin-rich domains at biomembranes, which demonstrated high affinity towards RGD moieties functionalized in the CMC-DOX-RGD and CMC-DOX-RGD-R bioconjugates. Therefore, this high affinity between tripeptide RGD and the αvβ3 integrin receptors in the extracellular matrix (ECM) of cancer cells has triggered the endocytosis via endo-lysosomal pathways, which were more pronounced due to the higher metabolism of cancer cells.23-25,88-90 These endo-lysosomal vesicles are responsible for trafficking and degradation of bioconjugates in the endocytic process, which resulted in the higher rate of DOX released into the cytosol and causing cell death. Conversely, normal HEK 293T cells showed much lower density of endo-lysosomal vesicles at fluorescent bioimaging analysis due to the lower content of αvβ3 integrin receptors at the membrane associated with a slower cellular metabolism. These results are consistent with the overall behavior verified by MTT and CVS discussed in previous sections (endorsed by mathematical model analysis of release profile), where HEK 293T cells showed higher cell viability responses towards polymer-drug-peptide conjugates while U-87 MG cancer cells were effectively killed. In summary, we demonstrated in this study that the biofunctionalization of CMC-DOX conjugates with RGD-R peptides showed affinity towards αvβ3 integrin receptors overexpressed by U87 cells, which also affected distinctly the endocytic pathways of normal and cancer cells regulating the release rate of the active anticancer drug. To this end, our results demonstrated that CMC-DOX-RGD-R conjugates showed specific cellular uptake mechanisms and DOX release rates, which preserved normal HEK 293T cells against drug toxicity while effectively killing U-87 MG cancer cells.



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2h

Figure 18 – Fluorescence images showing (a) green emission of LysoTracker®, (b) red emission of DOX, and (c) overlapped CLSM images of green and red emissions for (A) HEK 293T and (B) U-87 MG cells after 30 min and 2 h of incubation with CMC-DOX-RGD-R conjugate (scale bar = 10 µm). 40 ACS Paragon Plus Environment

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2.3.2.5. Drug-induced apoptosis assessed by bioimaging - Confocal laser scanning microscopy In this study, Figure 19 and Figure 20 show results of apoptotic cells determined by laser confocal fluorescent microscopy analysis with Annexin V- Alexa Fluor™ 488 staining after incubating HEK 293T and U-87 MG cells for 6 h with free DOX and all the CMC-DOX-based conjugates. It was clearly evidenced by fluorescent bioimaging that normal and cancer cells, HEK 293T and U-87 MG, respectively, presented apoptotic cells after incubation with free DOX and conjugates for 6 h. These results demonstrated the effective delivery of DOX by the polymer-DOX conjugated prodrugs with anticancer efficacy, which followed drug-induced apoptosis pathways leading to cell death. These findings are consistent with the literature where several in vitro studies suggested that polymerdoxorubicin conjugates function through molecular mechanisms based on stronger activation of

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apoptosis-signaling pathways.16,61

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Figure 19 – Fluorescence images of (a) Annexin V - Alexa Fluor™ 488 staining of apoptotic HEK 293T cells after incubation with DOX (A) and prodrug systems ((B) CMC-DOX, (C) CMC-DOXRGD and (D) CMC-DOX-RGD-R) for 6 h. (b) Fluorescence images of DOX and (c) Bright field + overlapped images of Annexin V- Alexa Fluor™ 488 and DOX (scale bar = 10 µm). 41 ACS Paragon Plus Environment

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(c) BF + Merge

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(a) AnnexinV-Alexa®488


Figure 20 – Fluorescence images of (a) Annexin V - Alexa Fluor™ 488 staining of apoptotic U-87 MG cells after incubation with DOX (A) and prodrug systems ((B) CMC-DOX, (C) CMC-DOXRGD and (D) CMC-DOX-RGD-R) for 6 h. (b) Fluorescence images of DOX and (c) Bright field + overlapped images of Annexin V - Alexa Fluor™ 488 and DOX (scale bar = 10 µm).

2.3.3. Chorioallantoic membrane (CAM) – in vivo assay The CAM assay has been widely accepted as an in vivo xenograft model for the evaluation of toxicity and activity of drug delivery systems that bridges the gap between basic in vitro studies and more complex animal models for cancer research. The CAM model is naturally immunodeficient and highly vascularized, making it an ideal system for studying cancer growth and tumor behavior. Additionally, the CAM contains extracellular matrix proteins (e.g., fibronectin, laminin, collagen, integrin alpha(v)beta(3), and MMP-2) making it a very interesting model to study tumor invasion and metastasis.91-93 Thus, in this study, CAM assay was used for drug activity evaluation by the effects on the vasculature of chorioallantoic membrane. DOX is a compound with antiangiogenic activity and the efficiency of prodrugs containing antiangiogenic drugs with the CAM model can be evaluated by 42 ACS Paragon Plus Environment

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the assessment of the occlusion of the blood vessels. Initially, the results (Figure 21) indicated that polymer conjugation retained the DOX antiangiogenic activity. At the higher concentration of DOX (25.0 µM) in the CMC-DOX-based systems, it was observed that about 15 % of embryos died and the embryos that survived showed significant antiangiogenic activity (% blood vessels ≤ 30 %). At lower concentration of DOX (2.5 µM), embryo’s death was not observed, and polymer-drug conjugates significantly reduced the capillary area when compared to negative control (PBS). More importantly, the higher antiangiogenic activity was verified for CMC-DOX-RGD-R conjugates. Inhibition of angiogenesis by arginine containing materials has been reported in literature. It has been associated with the interaction of arginine clusters with heparin sulfate proteoglycan (HSPG) binding site inhibiting the angiogenesis by preventing interaction of HSPG with angiogenic activators like vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-2), among others.91



(A)

2.5 µM 25.0 µM

100

Blood Vessels (%)

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80 60 40 20 0 - Control (PBS)

(B)

CMC-DOX


(a)

(b)

(c)

(d)

Figure 21 - (A) Capillary area in the CAM after application of DOX-loaded prodrugs and PBS (negative control). (B) Representative images of capillary area observed in the CAM ((a) PBS, (b) CMC-DOX, (c) CMC-DOX-RGD and (d) CMC-DOX-RGD-R).


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3. Conclusions In this study, we focused on the synthesis and comprehensive characterization of new polymer-drug systems based on the conjugation of carboxymethylcellulose (CMC, biopolymer), and doxorubicin (DOX, anticancer drug) and functionalized with peptides RGD and L-arginine (R) for prospective clinical applications in targeting chemotherapy of cancer cells. FTIR, UV-visible and PL spectroscopy techniques showed the covalent conjugation via the formation of amide bonds between carboxylic groups of carboxymethylcellulose and amino groups from doxorubicin, RGD and R (peptides). ZP and DLS measurements demonstrated the formation of charged nanoparticulate colloidal systems with hydrodynamic size ranging from approximately 30 to 90 nm depending on the pH and the chemical components of bioconjugate. These results were interpreted as the tridimensional conformation of these macromolecular superstructures caused by the balance of hydrophilic, hydrophobic and electrostatic interactions among the components and theirs chemical functional groups. The results of in vitro cell viability assays assessed by MTT and CVS methods demonstrated that free DOX and all the polymer-drug bioconjugates showed different cytotoxicity against cancer cell types (bone, breast and brain cancer cells, SAOS, MCF7 and U87, respectively) compared to normal embryonic cells (HEK 293T, reference). This was assigned to the combination physicochemical characteristics of bioconjugates with distinct cellular uptake pathways and endocytic mechanisms. Additional biological assays with fluorescent biomarkers using confocal laser microscopy endorsed these findings and evidenced the major mechanisms involved of DOX release from the polymer-drug systems in the cell compartments (i.e., endosome, lysosome, cytosol, nuclei) assigned to the cleavage of amide bonds from bioconjugates compared to free DOX. Moreover, the CMC-DOX conjugates dually biofunctionalized with integrin-target receptor tripeptide (RGD) and arginine (R) as cell-penetrating peptide (CMC-DOX-RGD-R) behaved as “smart” drug delivery systems (DDS) promoting remarkable reduction of the cytotoxicity towards normal cells (HEK 293T) while retaining high killing activity against cancer cells. These results were also analyzed using mathematical models, which showed the best fitted release profiles compatible with the diffusion controlled transport (i.e., Higuchi equation) for free DOX, CMC-DOX, and CMC-DOXRGD conjugates. Conversely, CMC-DOX-RGD-R conjugates showed unique drug release kinetics without matching the tested models, which supported the previous findings of anomalous behavior caused by conjugation of RGD and R moieties to the macromolecular structure affecting the cellular metabolism and kinetics. Therefore, innovative CMC-DOX-peptide nanocarriers were developed for simultaneously integrin-targeting and high killing efficacy against cancer cells, while preserving



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healthy cells from inherent cytotoxicity of anticancer drugs with promising use in cancer chemotherapy applications.

4. Experimental Procedure 4.1. Materials and cell cultures Carboxymethylcellulose sodium salt (CMC, degree of substitution, DS = 0.7; average molecular mass, Mw = 250,000 Da; molecular mass per unit = 214 g mol-1; degree of polymerization = 1,168; medium viscosity = 180 cps, 4 % in H2O at 25 ˚C), 2-(N-Morpholino)ethanesulfonic acid (MES, > 99 %, low moisture content), 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC, ≥ 98 %), doxorubicin hydrochloride (DOX, ≥ 98.0%), arginine-glycine-aspartic acid (Arg-Gly-Asp, RGD, ≥ 97 %), L-arginine (R, ≥ 98 %), ethalonamine hydrochloride (≥ 99.0 %), rhodamine 6G (suitable for fluorescence), 3-(4,5-dimethylthiazol-2yl) 2,5-diphenyltetrazolium bromide (MTT, > 98 %), Triton™ X-100, sodium dodecyl sulfate (SDS, ≥ 99.0 %), crystal violet (dye content ≥ 90 %, certified by the Biological Stain Commission), absolute ethanol (≥ 99.8 %), paraformaldehyde (95 %), and hydrochloric acid (HCl, 37 %) were purchased from Sigma-Aldrich (USA). Acetic acid glacial (≥ 99.7 %) was supplied by VETEC (Brazil). Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI 1640), fetal bovine serum (FBS), phosphate buffered saline (PBS), penicillin G sodium, streptomycin sulfate, and amphotericin-b were supplied by Gibco BRL (USA). Hydromount was purchased from Fisher Scientific Ltd. (USA). LysoTracker® Green DND-26, Annexin V-Alexa Fluor™ 488 conjugate and TO-PRO®-3 were supplied by InvitrogenTM (USA). Aforementioned chemicals were used without further purification, deionized water (DI water, Millipore SimplicityTM) with resistivity of 18 MΩ cm was used to prepare the solutions, and the procedures were performed at room temperature (RT, 23 ± 2 ˚C), unless specified otherwise. Human embryonic kidney (HEK 293T, American Type Culture Collection - ATCC® CRL-1573™) cells and immortalized human osteosarcoma-derived (SAOS, ATCC® HTB-85™) cells were cells were provided by Federal University of Minas Gerais/UFMG. Human brain likely glioblastoma (U87 MG, ATCC® HTB-14™) cells and human breast adenocarcinoma (MCF7, ATCC® HTB-22™) were purchased from Brazilian Cell Repository (Banco de Células do Rio de Janeiro: BCRJ, Brazil; cell line authentication molecular technique, Short Tandem Repeat (STR) DNA; quality assurance based on the international standard NBR ISO/IEC 17025:2005).


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4.2. Polymer-drug Bioconjugation The DOX anticancer drug, RGD tripeptide, and L-arginine were bioconjugated to the CMC polysaccharide backbone using 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) as a “zero-length” crosslinking agent in MES buffer (2-(N-Morpholino)ethanesulfonic acid, 0.25 M, pH 5.5 ± 0.1). CMC solution (2.0 wt. %) was prepared by adding sodium carboxymethylcellulose powder (CMC/Na, 2.0 g) to 100 mL of MES buffer and stirring at room temperature until complete solubilization occurred. EDC, RGD, L-arginine, and DOX were also dissolved in MES buffer. Polymer-drug bioconjugation of was performed as follows: 1.0 mL of EDC solution (12.5 wt %) was added to the reaction flask with 10 mL of CMC solution (2.0 wt. %) and magnetically stirred for 15 min at 6 ± 2 ˚C. Under continuous stirring, DOX solution (0.145 wt. %, 400 µL) was dropped into the flask, and the system was incubated at RT for 2 h in the dark. This prodrug system was referred to as “CMC-DOX” (1000:1, CMCunit:DOX molar ratio). For the preparation of the polymer-drug conjugate modified with RGD, EDC solution (0.6 wt. %, 1 mL) was added to 12 mL of “CMC-DOX” and magnetically stirred for 15 min at 6 ± 2 ˚C. Then, RGD solution (0.1 wt. %, 1.0 mL) was introduced into the flask, and the system was incubated at RT for 2 h in the dark under magnetic stirring. This prodrug system was referred to as “CMC-DOXRGD” (1000:1:15, CMCunit:DOX:RGD molar ratio). Lastly, CMC-DOX conjugate modified with RGD and L-arginine (referred to as “CMC-DOX-RGDR”) was prepared by adding L-arginine solution (4.28 wt. %, 5.0 mL) to 18 mL of “CMC-DOXRGD” solution and in incubating at RT for 2 h in the dark with magnetic stirring after activation of carboxylic groups of CMC with EDC solution (12.5 wt. %, 1 mL). Molar ratio CMCunit:DOX:RGD:R was 1000:1:15:1000. All of the samples were kept in the dark at RT overnight and, in the sequence, ethanolamine hydrochloride was added to the reaction flasks and magnetically stirred for 15 min at final concentration of 1.0 µM to quench the reaction. Then, the synthesized polymer-drug bioconjugates were dialyzed for 24 h (with water changes after 2 h and 4 h) in the dark against 2 L of distilled water using a Pur-A-Lyzer™ Mega Dialysis Kit (Sigma, cellulose membrane with molecular weight cutoff, MWCO, of 12,000 Da) under moderate stirring at RT. After purification, the prodrug solutions were stored at 6 ± 2 ˚C until further use. For evaluating the drug loading efficiency (DLE), prodrug systems (before dialysis) were centrifuged (15 min at 14,000 rpm and 4 ± 1 ˚C, Hettich Mikro 200R) using an ultracentrifuge filter with a cutoff cellulose membrane of 50,000 Da (Amicon filter, Sigma). The filtrate was collected and analyzed 47 ACS Paragon Plus Environment


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by UV–vis spectroscopy (Lambda EZ-210, Perkin Elmer) to determine the DOX concentration based on the Beer–Lambert correlation curve. The loading efficiency (DLE, %) was calculated using Equation 1. DLE = ((A-B)/A)* 100

(1)

where A (mg mL-1) is the initial concentration of DOX in solution and B (mg L-1) is the concentration of DOX at filtrate.

4.3. Physicochemical Characterization of Bioconjugates Fourier-transform infrared spectroscopy (FTIR) spectra of the films prepared from CMC polymer and prodrug solutions at pH 5.0 ± 0.2 were obtained using attenuated total reflectance method (ATR, 4000-675 cm-1, 32 scans, and 4 cm-1 resolution, Nicolet 6700, Thermo Fischer) with background subtraction. Ultraviolet-visible (UV-vis) spectroscopy measurements were performed (Perkin-Elmer Inc., equipment Lambda EZ-210, USA)) in transmission mode with samples in a quartz cuvette over the wavelength range between 600 and 350 nm. All of the experiments were conducted in triplicate (n = 3) unless specifically noted. Photoluminescence spectroscopy (PL) was performed based on spectra acquired at RT using a violet diode laser module at 405 nm excitation wavelength (λexc) (150-mW, Roithner LaserTechnik, Germany) coupled to a USB4000 VIS-NIR (visible-near infrared) spectrophotometer (Ocean Optics, Inc., USA). All of the tests were performed using a minimum of four repetitions (n ≥ 4). Quantum yield (QY) was measured according to the procedure using Rhodamine 6G in ethanol as the standard at λexc = 405 nm.94 Dynamic light scattering (DLS) and zeta potential (ZP or ζ-potential) analyses were performed using ZetaPlus instrument (Brookhaven Instruments Corporation, 35 mW red diode laser light, wavelength λ=660 nm) with a minimum of ten replicates. The ZP measurements were performed at 25 ± 2 ˚C under the Smoluchowski approximation method with a minimum of ten replicates (n ≥ 10). TEM (Transmission Electron Microscopy) images were obtained using Tecnai G2-20 (FEI Company at 200 kV,) microscope after drying the sample (CMC-DOX dissolved in PBS) onto carbon-coated copper grids and removal of buffer salts gently dipping in DI water (without coating or staining). In vitro (acellular) drug release was performed at pH 7.4 and pH 5.5 (triplicates) by dialysis method. Briefly, 10 mL DOX-loaded prodrugs (DOX concentration 25.0 µM) or free DOX solution (25.0 µM) were placed into dialysis bag (MWCO 12,000 Da, Sigma-Aldrich) and tightly sealed. Then the

dialysis bags were immersed into 100 mL of aqueous release medium (pH 5.5 ± 0.2 or pH 7.4 ± 0.2) 48 ACS Paragon Plus Environment

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at 37 ± 1 oC with constant shaking (50 ± 3 rpm) for 7 days. At determined time intervals, 2 mL of release medium was collected and analyzed by UV-vis to determine the concentration of dissociated DOX based on the Beer–Lambert correlation curve (λ = 484 nm) and calculate the cumulative release rate.

4.4. Biological Characterization of Bioconjugates All of the biological tests were conducted according to ISO 10993-5:2009/(R)2014 (Biological evaluation of medical devices: Tests for in vitro cytotoxicity) using kidney cell line of a human embryonic culture (HEK 293T), malignant glioma cells (U-87 MG), immortalized human osteosarcoma-derived (SAOS), and human breast adenocarcinoma (MCF7). HEK 293T (passages 18, 35, 60, and 63), U-87 MG (passage 21, 39, 42, and 45) and SAOS (passages 38 and 42) cells were cultured in DMEM with 10 % FBS, penicillin G sodium (10 units mL-1), streptomycin sulfate (10 mg mL-1), and amphotericin-b (0.025 mg mL-1) in a humidified atmosphere of 5% CO2 at 37 ˚C. MCF7 (passages 28 and 29) cells were cultured in RPMI 1640 with the same supplements and conditions.

4.4.1. Cell viability assays in vitro For cell viability assays, control samples were designed as follows: control group (cell culture with DMEM or RPMI 1640 medium and 10 % FBS); positive control (cell culture with DMEM or RPMI 1640, 10 % FBS and 1.0 % v/v Triton™ X-100); and negative control (cell culture with DMEM or RPMI 1640 medium, 10 % FBS and chips of sterile polypropylene Eppendorf®, 1 mg mL−1, Eppendorf, Germany). The percentage of cell viability was calculated after blank corrections, according to Equation 2, where the values of the control group (i.e., wells with cells but no samples) were set to 100 % cell viability.

Cell viability = (Absorbance of sample and cells)/(Absorbance of control) × 100 %

(2)

4.4.1.1. Mitochondrial activity (MTT) assay MTT (3-(4,5-dimethylthiazol-2yl) 2,5-diphenyl tetrazolium bromide) experiments were performed to evaluate the toxicity of free-DOX and prodrug conjugates. All of the cells were plated (1×104 cells/well) in 96-well plates. Cell populations were synchronized in serum-free media for 24 h. After that, the media volume was suctioned and replaced with DMEM or RPMI 1640 media containing 10 % FBS for 24 h. Then DOX and prodrug solutions were added to individual wells at final concentrations of 0.1 µM, 1.0 µM, 10.0 µM, or 50.0 µM of DOX. After 24 h, all media were 49 ACS Paragon Plus Environment


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aspirated and replaced with 60 µL of culture media containing serum to each well and images of cells were acquired on an Leica DMIL LED (Germany) inverted microscope. Then 50 µL of MTT (5 mg mL−1) was added to each well and incubated for 4 h in an oven at 37 ˚C and 5 % CO2. Next, 40 µL SDS solution/4 % HCl was placed in each well and incubated for 16 h in an oven at 37 ˚C and 5 % CO2. Then, 100 µL were removed from each well and transferred to a 96-well plate. The absorbance was measured on iMark™ Microplate Absorbance Reader (Bio-Rad) with a wavelength filter at λ=595 nm and cell viability was calculated using Equation 2.

4.4.1.2. DNA staining assay (Crystal Violet Staining, CVS) Crystal violet staining (CVS) cell cytotoxicity assay was used to assess the cytotoxicity of DOX and CMC-DOX bioconjugates. Cells were plated, synchronized and prepared as previously described in Section 2.4.1.1. Then, cells were incubated with free DOX and prodrug solutions at final concentrations of 0.1 µM, 1.0 µM and 10.0 µM of DOX for 6 h or 24 h. Then cells were washed with PBS (1 % v/v), fixed with ethanol (70 % v/v) and stained with violet crystal (0.5 wt % in ethanol). Stained cells were solubilized in acetic acid (10 % v/v) and absorbance at λ = 610 nm was measured using plate reader (Multiskan Go, Thermo-Scientific). Cell viability was measured according to Equation 2.

4.4.2. Cellular uptake of polymer-drug bioconjugates - Confocal laser scanning microscopy (CLSM) HEK 293T and U-87 MG cells were plated (5×105 cells per well) in 6-well plate. The cells were incubated for 4 days in 5 % CO2 at 37 ˚C and synchronized for 24 h. Then, 500 µL of DOX solution and DOX prodrug solutions were added to each well at DOX dosage of 10.0 µM. For the reference control, cells were incubated only with the DMEM medium with 10 % FBS. After incubation at 37 ˚C and 5 % CO2 from 15 min up to 360 min (depending on de experiment), the cells were washed with PBS. In the sequence, the cells were fixed with paraformaldehyde (4 % v/v in PBS) for 30 min, washed three times with PBS, and cover slips were mounted with Hydromount®. Images were obtained with a Zeiss LSM Meta 510 confocal microscope (Carl Zeiss, Germany) using the water immersion

(objective

63×

Plan-Apo/1.4

NA,

Numerical Aperture).

For green-emission

(LysoTracker® Green and Alexa Fluor™ 488), argon laser was used to excite at λexc = 488 nm and emission was collected at 505-550 nm. For red-emitting DOX, excitation was at λexc = 568 nm and emission was collected at LP 585 nm (LP = low pass). Excitation at λ = 633 nm and observation using LP 650 nm was selected to detect far-red fluorescence of TO-PRO®-3 and the image obtained 50 ACS Paragon Plus Environment

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for this channel was presented in blue color. Dual-color confocal fluorescence images were recorded separately in the correspondent channel and merged afterwards.

4.4.2.1. Cellular uptake of CMC-DOX bioconjugates - Internalization and kinetics analyses To verify intracellular uptake, DOX drug and polymer-drug conjugates were monitored using confocal laser microscopy after treatment of HEK 293T and U-87 MG cells for 15 min, 30 min, 60 min, 2 h and 6 h, using the inherent fluorescence imaging capability of DOX to obtain information on drug distribution inside the cells. Plot of intensity profiles and measurements of mean fluorescence intensities were performed using public domain image processing software (ImageJ software, version 1.50). 4.4.2.2. Cellular uptake of CMC-DOX bioconjugates - DNA-staining with TO-PRO®-3 To selective staining the nuclei of the HEK 293T and U-87 MG cells, after incubation with DOX and prodrug conjugates for 30 min, 2 h and 6 h, they were washed and treated with TO-PRO®-3 for 30 min according to the manufacturer's protocol.

4.4.2.3. Cellular uptake of CMC-DOX bioconjugates - Lysosomal biomarkers HEK 293T and U-87 MG cells after incubation with CMC-DOX-RGD-R conjugates at for 30 min and 120 min (2 h) were washed and additionally stained for 2 h with 50 nM of lysosome dye (LysoTracker® Green DND-26) according to the manufacturer's protocol.

4.4.2.4. Drug-induced apoptosis assessed by bioimaging - Confocal laser scanning microscopy (CLSM) HEK 293T and U-87 MG cells after incubation with DOX and polymer-drug conjugates for 6 h were washed and additionally stained with a green fluorescent annexin V conjugate (Annexin V - Alexa Fluor™ 488), according to the manufacturer's protocol, as an indicator of apoptosis.

4.4.3. Chorioallantoic membrane (CAM) – in vivo assay Fertilized chicken eggs (n = 25 for each group) were placed in a hatching incubator maintained at 37 °C and 60 % humidity. Three days after fertilization, a hole of approximately 1 cm in diameter was made in the eggshell to provide access to the chorioallantoic membrane (CAM). Five days after fertilization, samples of prodrug solutions (50 µL, 2.5 µM or 25.0 µM) were applied over the CAM surface in a well-defined place, which was marked with a cellulose disk. After 48 h, the CAM 51 ACS Paragon Plus Environment


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surrounding the cellulose disk was removed from each egg and analyzed with a light microscope (Leica, model DM4000B) coupled to a Leica digital CCD camera model DFC 280 (Software Leica Application Suite V 3.3.0). The images were used for the quantification of the capillary area using the image processing software (ImageJ, v. 1.44p). For control, the same procedure was performed and 50 µL of PBS at pH 7.4 (negative control) was applied over the CAM surface. The negative control group was fixed as 100% for the calculation of the capillary area changes. Data were presented as mean ± standard error.

4.4.4. Statistical analysis Statistical significance was tested using One-way ANOVA followed by Bonferroni’s method. At α confidence level value α < 0.05 was considered statistically significant. The experiments were performed at least in triplicate (n ≥ 3).

Acknowledgments The authors acknowledge the financial support from the Brazilian research agencies (CNPq, CAPES, FAPEMIG and FINEP. Finally, the authors thank the staff at the Center of Nanoscience, Nanotechnology and Innovation-CeNano2I/CEMUCASI/UFMG for the spectroscopy analyses.

Funding Sources CNPq

(140810/2015-3;

PQ1B–306306/2014-0;

UNIVERSAL-457537/2014-0;

PIBIC-

2016/2017/2018), CAPES (PROEX-2010-2017; PNPD; PROINFRA2010-2014), FAPEMIG (PPM00760-16), and FINEP (CTINFRA-PROINFRA 2008/2010/2011).

Supporting Information. Additional figures are presented as described in the manuscript.

Author Contributions The manuscript was written through equally contributions from all authors. All authors have read and given approval to the final version of the manuscript.

Conflicts of Interest The authors declare that they have no competing interests.


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Table of Contents

Extracellular fluid

Prodrug conjugate Cell membrane O

Amide bond HO

Endosome

Lysosome

Nucleus

H3C

O HN

HO

O

OH

O

HO

O CH3

HO

HO

O

O

O HO

NH

HO

OH

O

O Na

NH

NH

O

O

O

NH

Amide bond

O O O

O

O

HO

OH

O O

O

DOX Drug

O

O

CMC backbone

OH

HO

O

NH

O

Amide bond

HN

NH2

Integrin target

O OH

RGD

H2N O HN

Penetrating peptide

O NH

Arginine (R)

O

O OH HO

Polymer-Drug-Peptide Bioconjugates – Biopolymer-based chemotherapeutics for specific targeting cancer cells.



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Design and Development of Polysaccharide-Doxorubicin-Peptide Bioconjugates for Dual Synergistic Effects of Integrin-targeted and Cell-penetrating Peptides for Cancer Chemotherapy Alexandra A. P. Mansur†, Sandhra M. Carvalho†,§,#, Zélia I.P. Lobato§, Maria de Fátima Leite#, Armando da Silva Cunha Jr‡, Herman S. Mansur†,

Table of Contents

Extracellular fluid

Prodrug conjugate Cell membrane O

Amide bond HO

Endosome

H3C

O HN

HO

OH

O

O CH3

HO

HO

O

O

O HO

NH

HO

OH

OH

O

O Na

NH

NH

O

O

O

NH

Amide bond

O O O

O

O

HO

OH

O O HO

DOX Drug

O

O

Nucleus

O

HO

CMC backbone

Lysosome

OH

HO

O

NH

O

Amide bond

HN

NH2

Integrin target

O OH

H2N

RGD

O HN

Penetrating peptide

O NH

Arginine (R)

O

O OH HO

Polymer-Drug-Peptide Bioconjugates – Biopolymer-based chemotherapeutics for specific targeting cancer cells.

 To whom correspondence should be addressed: Federal University of Minas Gerais, Av. Antônio Carlos, 6627 – Escola de Engenharia, Bloco 2 – Sala 2233, 31.270-901, Belo Horizonte/MG, Brazil; Tel: +55-31-34091843; Fax: +55-31-34091843; E-mail: [email protected] (H. Mansur)


Design and Development of Polysaccharide-Doxorubicin-Peptide

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