Fatty-Amine-Conjugated Cationic Bovine Serum Albumin

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Fatty-Amine-Conjugated Cationic Bovine Serum Albumin Nanoparticles for Target-Specific Hydrophobic Drug Delivery Abhishek Saha, Nirmalya Pradhan, Soumya Chatterjee, Rakesh Singh, Vishal Trivedi, Arindam Bhattacharyya, and Debasis Manna ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.9b00607 • Publication Date (Web): 08 May 2019 Downloaded from http://pubs.acs.org on May 9, 2019

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ACS Applied Nano Materials

Fatty-Amine-Conjugated Cationic Bovine Serum Albumin Nanoparticles for Target-Specific Hydrophobic Drug Delivery Abhishek Saha,§,† Nirmalya Pradhan,§,† Soumya Chatterjee,ǁ Rakesh Singh,‡ Vishal Trivedi,*,‡ Arindam Bhattacharyya,ǁ and Debasis Manna*,† †

Department of Chemistry, and ‡Department of Bioscience and Bioengineering, Indian Institute

of Technology Guwahati 781039, Assam, India. ǁ

Department of Zoology, University of Calcutta, Kolkata 700019, West Bengal, India.

KEYWORDS: Anticancer activity • cationic BSA nanoparticles • fatty amine conjugation • sustained release • target specific drug delivery

ABSTRACT

Protein based nanostructures has reformed the nanoscience and nanotechnology on the account of their smaller sizes and greater surface areas, which instigates their interactions with other molecules. The protein nanoparticles (NPs) have better biocompatibility, biodegradability and also have the easy access for additional surface modifications. These NPs have been successfully

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used as drug delivery system with increased bioavailability and reduced toxic side effects of the drug molecules. Herein, we report a simple approach to formulate fatty-amine-conjugated cationic BSA (FCBSA) NPs by conjugating laurylamines to the BSA protein. Partial neutralization of the negatively charged glutamic acid or aspartic acid residues by the formation of amide bond with laurylamines leads to the formation of cationic NPs under physiological conditions (isoelectric point = 7.7 and ζ = +7 mV at pH 7.2). The NPs exhibit high stability against thermal, pH, and proteolytic enzyme stresses. The NPs demonstrated excellent biocompatibility against both normal and cancer cell lines. The protein NPs efficiently encapsulate hydrophobic anticancer drug, doxorubicin (Dox) and shows controlled release property (~ 40% release after 3 days), human blood serum stability, antifouling property and higher binding affinity for the anionic membranes. The biotin tagged cationic FCBSA (btFCBSA) also showed very similar biophysical properties as of only FCBSA. Furthermore, the cellular studies also showed that bt-FCBSA can efficiently deliver Dox to the biotin receptorpositive HeLa cells leading to significant cell death. In vivo assessment of the Dox encapsulated bt-FCBSA on Ehrlich ascites carcinoma cells bearing female Swiss albino mice revealed significant inhibition of tumor growth. Overall, an easy access to the fatty amines modified cationic protein NPs with their surface modification capabilities could be eminent candidates for both passive and active targeted delivery of anticancer agents.

INTRODUCTION Cancer has emerged as one of the most life threatening diseases throughout the world today.1-3 Although significant progress has been made in the past two decades in developing anticancer drugs, still a great deal of work to be done, explicitly in the field of early diagnosis and targeted


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drug delivery.1-2, 4 Chemotherapy is one of the prevalent and effective approaches to treat cancer tissues in the patient’s body.2-3 However, the major drawbacks of the chemotherapeutic agents are poor aqueous solubility, chemical instability and high toxicity against normal cells and eventually development of multidrug resistance.5-6 Unfortunately, the vast majority of the chemotherapeutic agents either FDA-approved or under development stages exhibit poor aqueous solubility. Various approaches, such as salification of ionizable drugs, complexation with cyclodextrins and other others have shown some significance in overcoming the solubility problem.7-10 However, most of these strategies failed to augment the therapeutic benefit in drug treatment. The burst or dormant release, tissue specificity, excretion and others could be the other reasons for the suboptimal performance of the above formulations. The doxorubicin (Dox) is one of the most widely used hydrophobic chemotherapy drugs. However, the use of Dox is associated with unalterable cardiac toxicity, other side effects and drug resistance.5-9, 11 The development of suitable drug delivery systems (DDS) can improve the efficacy of chemotherapeutic approaches by optimizing the drug encapsulation, distribution, and minimizing the side effects due to restricted delivery to healthy cells.2-3 Currently, liposomes, micelles, polymers, nanoparticles (NPs) and other drug delivery vehicles with or without cancer cell targeting ligands are being commonly used to prolong and maintain efficient plasma concentration of the anticancer agents and maximize site-specific delivery.2, 12-13 Modification of the surface of DDS with cancer cell specific ligand is known to improve their targeting ability and also improved the pharmacokinetics, biodistribution profile and protects the anticancer drug molecules from premature metabolism or degradation.3, 14 The target-specific nanovehicles are considered as one of the mainstream DDS for cancer treatment, because of the presence of highly disorganized vasculature and defective lymphatic drainage systems of the cancerous cells. The


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enhanced permeation and retention (EPR) effect allows the intravenously injected nanomedicines to extravasate from the blood vessels and accumulate within tumor tissues.2, 4, 15 In this regard, the NPs-based DDS have been widely investigated for the development of safer with moreeffective therapeutic modalities for the delivery of hydrophobic drug molecules. Currently, various natural or synthetic materials containing NPs-based drug formulations like Doxil, DepoCyt, Oncaspar, Genexol-PM, CAELYXTM and others are either FDA-approved or under clinical trials for cancer therapy.16-17 However, most of these reported NPs-based drugs have numerous limitations, including, biosafety and targeted delivery. The therapeutic agents get released in the perivascular cells of tumor and the NPs get accumulated in the reticuloendothelial cells of liver and spleen. These pitfalls of the existing NPs-based DDS encouraged the scientists to develop biocompatible material-based DDS that should have better stability in plasma, reduced cytotoxicity, targeted delivery with proficient loading and controlled release aptitude.2, 15, 18-19

The choice of suitable biocompatible platform is crucial, because it directly control the

drug delivery efficacy and degree of functionality that can be attached to its surface for targeted delivery.2-3, 20 Plasma protein like albumin has several favorable features including, biocompatibility, biodegradability, environmental sustainability, cost effectiveness, non-immunogenicity, long half-life and lyoprotectant to formulate successful DDS.13,

21-22

Fortunately, the albumin NPs-

based DDS also have not been observed to significantly affect the stability or physicochemical properties of the encapsulated drug molecules. However, the albumin itself has two major drawbacks- adsorption of other serum proteins and limited ability to deliver hydrophobic drug molecules.2 Any drug, even if administered intravenously, orally, subcutaneous, sublingual or intramuscularly, is transported through the blood circulation systems and interacts not only with


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the low-molecular weight compounds and various cellular components but also with various plasma proteins.3 The adsorption of serum proteins can potentially destabilize most of the DDSs, leading to a premature release of encapsulated drug molecules in the blood that could promote the side effects.1-3 A successful application of albumin in the formulation of Abraxane, a paclitaxel bound albumin NPs, to treat different types of cancers has inspired researchers in exploring the formulation of other albumin based DDS.2, 4 Abraxane reduces the vehicle-related side effects, but it has poor colloidal stability in blood.4 Construction of a biocompatible shell for the micelles, NPs and liposomes is considered as one of the most advanced and popular strategies to get stability in the plasma. In addition, cationic biocompatible surface offers salient features like efficient electrostatic interaction with the anionic lipids (like phosphatidyl serine), which are highly abundant at the outer surface of the plasma membranes of the cancer cells in comparison with the normal cells.23-25 It also provides an excellent antifouling property to the carrier, allowing them to circulate freely in the plasma and accumulate at a specific site via passive targeting.26 The vitamin biotin (vitamin B7 or H) is one of the well-known cancer cell‐ targeting ligands and has the potential to target and bind the biotin-transporters–overexpressing at the surfaces of the tumor cells.27-28 Hence, biotin modified albumin NPs could successfully deliver the hydrophobic anticancer drugs to targeted tumor cells. In the present study, BSA-based novel drug delivery vehicle was formulated. The native BSA protein contains 99 amino acids with carboxylic acid containing side chain and 87 amino acids with amine-containing side chain, suggesting an overall anionic surface potential.29 We hypothesized that the modification of the amino acids with carboxylic acid containing side chain of the BSA protein using fatty amines would self-assembled to form NPs having hydrophobic core and cationic surface in aqueous medium. In this regard, the fatty amines conjugated cationic


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BSA (FCBSA) NPs were synthesized by amide coupling method for low cost, rapid and effective drug delivery applicability (Figure 1). Additional cysteine labeling with biotin

Figure 1. Schematic representation of the synthesis of fatty amine conjugated cationic BSA nanovehicles formulation, its surface modification with biotin, capacity for anti-biofouling, and successful encapsulation and delivery of the anticancer drug Dox to biotin-receptor positive cancer cells.

derivative of FCBSA (bt-FCBSA) showed targeted delivery to the biotin-receptor positive cancer cells. This biotin labeled cationic nanovehicle efficiently encapsulated the Dox, increased the


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biosafety of Dox molecules, showed anti-fouling property and showed a slow and sustained Dox release profile in the physiological pH and burst release in acidic medium. The cytotoxicity and cellular uptake efficiency of the Dox loaded nanovehicle in biotin-receptor overexpressing cancer cell line indicate its target-specific deliverability. In addition to the in vitro studies, the Dox encapsulated nanovehicle was tested for efficacy by investigating the in vivo activities using Ehrlich ascites carcinoma (EAC) cells bearing female Swiss albino mice.

RESULT and DISCUSSION Preparation and Characterization of Fatty-Amine-Conjugated Cationic BSA ─ The laurylamine conjugated cationic BSA (FCBSA) and biotin-tagged laurylamine conjugated cationic BSA (bt-FCBSA) molecules were rapidly prepared by following standard reaction method of amide coupling and thiol-ene Michael addition reaction, respectively.30 The unfolding of BSA in DMSO solution that exposes its residues including acid group containing amino acids facilitated the conjugation of laurylamine with BSA protein. The free cysteine residues of FCBSA were modified with biotin-maleimide at physiological pH to produce the bt-FCBSA. The FCBSA and bt-FCBSA was initially investigated by analyzing the FTIR spectra (Figure 2A). The suppression of C-O-H in plane bending at 1395 cm-1 (for carboxylic acid functionality) and evolution of C-N stretching at 1117 cm-1 indicates the formation of amide bond between the carboxylic acid of BSA protein and evaluation of C-H stretching at 2971 cm-1 indicates the introduction of fatty amines. The presence of following characteristic peaks 3355 cm -1, 1707 cm1

, 1463 cm-1, 1264 cm-1, 1023 cm-1, 650 cm-1 in the bt-FCBSA spectra and further evaluation of

C-H stretching at 2971 cm-1 conforms biotin conjugation to FCBSA. Additional MALDI-TOF mass spectrometric analysis was performed to confirm the formation of FCBSA and bt-


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FCBSA.19 The MALDI-TOF intensity peak shows an increase in the average molecular weight from 66,340 to 74,837 Da due to the conjugation of fatty amines to BSA protein (Figure 2B). The change in mass spectra demonstrate that approximately 50 molecules of laurylamines were

Figure 2. Representative FTIR (A) and MALDI-TOF (B) spectra of BSA (─), FCBSA (─) and bt-FCBSA (─).

conjugated to each BSA molecule, indicating an average of 50% of the total carboxylic acid groups (99) of glutamic acid and aspartic acid residues of the BSA protein were conjugated with


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the laurylamine. An increase in the average molecular weight from 74,837 to 82,495 Da due to the conjugation of biotin-maleimide to FCBSA confirms addition of approximately 18 biotin molecules. To investigate the structural change due to the conjugation of fatty amines and biotin derivative, the circular dichroism (CD) analysis was performed. The results showed a significant change of secondary structural components of BSA protein in FCBSA and bt-FCBSA with complete loss of α-helix and ß-sheet motifs, suggesting a complete conformational change of the protein due to the conjugation of laurylamines to BSA and consequent addition of biotin derivative to FCBSA (Figure 3A and Table S1). The Trp-fluorescence measurements of BSA, FCBSA and bt-FCBSA revealed a significant structural change of the protein in FCBSA and btFCBSA (Figure 3B) and this observation is in complete agreement with the CD analysis. To investigate the overall structural arrangements of FCBSA and bt-FCBSA in aqueous solution, electron microscopic and atomic force microscopic analyses were performed. We hypothesize that the conjugation of laurylamine could increase the hydrophobicity and make BSA more amphiphilic, thus allowing them to form NPs in aqueous medium.18 The aqueous solution of FCBSA was prepared in 10 mM PBS (phosphate buffered saline) at pH 7.4. The analysis of collected TEM and FESEM images revealed that FCBSA and bt-FCBSA form distorted spherical NPs in aqueous solution with hydrodynamic diameter (dH) of < 200 nm (Figure 3C-D and Figure S2-3).31 The synthesized FCBSA or bt-FCBSA contains a large number of hydrocarbon long chains, which make the FCBSA or bt-FCBSA as amphiphilic molecule. The hydrophobic effect allow the amphiphilic molecules to arrange themselves in such a way that the hydrophilic groups remain close to the water interface, while the hydrophobic groups collapse together into the core


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to form spherical aggregates in the aqueous solution. The formation of NPs with hydrophobic core structures was also investigated by fluorescence spectroscopic analysis using pyrene, a hydrophobic probe.32 An increase in pyrene fluorescence intensity with the increase in

Figure 3. Characterization of the synthesized FCBSA and bt-FCBSA NPs. Representative CD spectra of BSA, FCBSA and bt-FCBSA in 10 mM PBS at pH 7.4 (A). The inset shows the secondary structural components (only α-helices and β-sheets) of the BSA, FCBSA and btFCBSA. Comparative Trp fluorescence spectra of BSA, FCBSA and bt-FCBSA (B).The TEM (C) and FESEM (D) images of bt-FCBSA. The CAC values of FCBSA and bt-FCBSA (E).Analysis of particle size distribution by DLS measurements for BSA, FCBSA and bt-FCBSA (F).


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FCBSA or bt-FCBSA concentrations suggest the formation of hydrophobic environment in the aqueous solution or incorporation of pyrene molecules with the hydrophobic region. The variation of I1/I3 fluorescence signals of pyrene at different concentrations of the FCBSA or btFCBSA was used to measure the critical aggregation concentration (CAC) of the fatty-amineconjugated BSA samples (Figure 3E and Figure S4). The calculated CAC values of the FCBSA and bt-FCBSA were 19 × 10−3 and 14 × 10−3 mg/mL, respectively. The low CAC values confirm the formation of soluble aggregates of FCBSA and bt-FCBSA in aqueous solution. The dynamic light scattering (DLS) measurements showed that the mean hydrodynamic diameters of the NPs from FCBSA and bt-FCBSA were within 178-187 nm.31 However, the mean dH of BSA was 50 nm under the similar experimental conditions. This difference in dH values indicates that the conjugated hydrophobic fatty amines contributed to the bigger size of FCBSA and bt-FCBSA aggregates (Figure 3F). The variation in the dH values of the nanovehicles could be due to the extent of fatty-amine-conjugation. Meanwhile, the surface potential (ζ) of BSA, FCBSA and btFCBSA were 22 ± 3, 7 ± 2 and 8 ± 2, respectively. The positive ζ values of FCBSA could be due to the conjugation of fatty amines with the glutamic and aspartic acids. The pH-dependent surface potential measurements revealed that the isoelectric point (IEP) of BSA, FCBSA and btFCBSA were 4.9, 7.8 and 7.9, respectively (Figure S5).33 This IEP value of FCBSA and btFCBSA suggests that at the physiological condition, its overall surface charge is cationic. This low positive surface potential value of the synthesized NPs could be beneficial, because higher positive surface potential may increase its cytolytic and cytotoxic behaviors.23, 34 The NPs with very high positive surface potential have the propensity to interact non-specifically with various negatively charged proteins in the blood circulation system leading to the adsorption of opsonins on its surface.35 The opsonized NPs can cause blood clotting and rapidly get cleared from the


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blood circulation system via the reticuloendothelial system.36 The in vivo studies of only stearylamine containing cationic liposome showed high toxicity due to the hemolysis of the erythrocytes.37 Several reports demonstrated that NPs with moderate cationic surface usually get internalized by clustering on anionic surfaces that are predominantly present on the cancer cells.38 To investigate the surface hydrophobicity of the FCBSA in aqueous solution fluorescence measurements were performed with ANS, a hydrophobic polarity sensitive fluorescent dye.14 The ANS dye shows characteristic high fluorescence intensity upon binding with the hydrophobic residues present on the protein surface. Whereas, the free ANS dye shows negligible fluorescence signals in polar medium. The ANS fluorescence intensity differences were investigated at 70 µM concentration (EC50 = 65 µM). Comparison of ANS fluorescence intensities in the presence of BSA, FCBSA and bt-FCBSA demonstrates a significant drop in protein-bound ANS fluorescence signal for FCBSA and bt-FCBSA compared to BSA (Figure S6). This reduction of protein-bound ANS fluorescence intensity suggests the formation of lower hydrophobic surface for FCBSA and bt-FCBSA. Introduction of laurylamines enhance the local hydrophobicity to BSA protein and promote additional amphiphilicity. The ANS fluorescence intensity for bt-FCBSA was slightly higher in comparison with FCBSA, suggesting the surface exposure of the biotinylated cysteine residues. The conformational changes in fatty-amineconjugated samples (as observed in CD measurements) also unmasked the polar amino acids to the surface. The lower hydrophobic surface could assist the fatty-amine-conjugated samples to flow smoothly in the aqueous medium. The spherical shape of the carriers also provides several advantages including lower hindrance against flow and higher membrane penetration capability.


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Stability of Fatty-Amines-Conjugated Cationic BSA ─The stability of nanocarriers in the biological media is one of essential parameters for their intended biological applications. Hence the stability of FCBSA and bt-FCBSA against thermal, pH, and proteolytic enzyme stresses were investigated.14 The thermal stability of FCBSA and bt-FCBSA in aqueous solution was examined across a temperature range of 20−80 °C by Trp and ANS fluorescence measurements (Figure 4A-D). A sharp decrease in temperature-dependent Trp-fluorescence signal (λem = 345 nm) was observed for BSA. This suggests an enhancement in polarity of the environment around Trp residues for BSA protein. However, it is important to know that there is already a huge difference in Trp-fluorescence between BSA and FCBSA even at 20 °C, which could be due to the exposure of maximum Trp resides to the aqueous environment (Figure 3B). The temperaturedependent Trp-fluorescence signals of FCBSA and bt-FCBSA were comparatively smaller but slightly increases with temperature, indicating an increase in hydrophobic environment around Trp residues (Figure 4C and S8).14 The temperature-dependent ANS fluorescence measurements also showed very similar change in signal strength for both BSA and FCBSA (Figure 4B). The ANS fluorescence measurements showed the reduction of protein-bound ANS fluorescence intensity (λem = 495 nm) with increase in temperature for BSA protein, indicating sharp decrease of surface hydrophobicity with temperature (Figure 4A and S7).14 Whereas, the protein-bound ANS fluorescence of FCBSA or bt-FCBSA was lower in comparison with that of BSA, but it slightly increases with temperature suggesting the higher thermal stability of FCBSA. The formation of NPs with cationic surface could be reason for low Trp and protein-bound ANS fluorescence signals of FCBSA. Hence, the increase in slight hydrophobic surface characters directly correlates with the stability of FCBSA against thermal stress.


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The stability of BSA, FCBSA and bt-FCBSA in different buffer solutions with pH 5-10 was investigated by fluorescence measurements. A sharp decrease in the protein-bound ANS fluorescence intensity for BSA protein was observed. However, the FCBSA-bound ANS

Figure 4. Stability of the synthesized FCBSA and bt-FCBSANPs against thermal, pH, and proteolytic enzyme stresses. Relative ANS-fluorescence spectra at 20 °C (A) and change in ANS-fluorescence signal at different temperatures (B). Change in Trp-fluorescence signal at different temperatures (C). Change in Trp-fluorescence signal at different pH (D). HPLC traces of BSA, FCBSA and bt-FCBSA in the absence and presence of proteinase K (PK) enzyme (E).

fluorescence intensity remained unchanged. The Trp fluorescence measurements (within the pH range of 4-10) showed higher stability for FCBSA and bt-FCBSA in comparison with the BSA


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under the similar experimental conditions (Figure 4D and S9). The sharp decrease in Trp fluorescence for BSA protein with an increase in the pH values of the solution suggests conformation changes and exposure of Trp residues to the aqueous environment. The pH dependent stability studies suggest the higher stability of FCBSA and bt-FCBSA than BSA. To investigate the stability of BSA, FCBSA, and bt-FCBSA against digestive enzymes, HPLC analysis was performed in the absence and presence of protease enzyme. Proteinase K (PK) is a serineprotease enzyme and it generally hydrolyses the native protein at a faster rate than the nonnative protein.39 The HPLC analysis showed that the PK enzyme successfully digests BSA protein, but failed to hydrolyze the FCBSA and bt-FCBSA NPs efficiently. The HPLC peak position of FCBSA (at RT = 21 minute) and bt-FCBSA (at RT = 29 minute) remained almost unchanged even in the presence of PK enzyme (Figure 4E). This increased stability of FCBSA and bt-FCBSA against PK digestion could be due the denatured and unfolded structure of the protein present in FCBSA and bt-FCBSA and inaccessibility of the digestion sites to the PK enzyme. Overall these results signify the synthesis of small and spherical FCBSA and bt-FCBSA NPs with high stability in aqueous solution. The photographs at different days also showed the dispersion property of bt-FCBSA in PBS buffer, pH 7.4, suggesting the stability of the nanovehicle (Figure S10).

Drug Loading and Release Profile ─ The ability of the BSA, FCBSA, and bt-FCBSA to encapsulate and deliver chemotherapeutic drugs and finally kill the cancer cells was investigated under in vitro conditions using model hydrophobic Dox. The desalting of commercially available Dox.HCl was performed according to the reported method.31,

40-41

The Dox encapsulation

efficiencies of BSA, FCBSA and bt-FCBSA were examined by UV-Vis spectroscopy (λex = 487


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nm) at pH 4.5 and 7.4. The active Dox-loading aptitude of the FCBSA (Dox to FCBSA ratio = 1: 5 (w/w)) was measured to be 27% and 47% at pH 4.5 and 7.4, respectively (Figure 5A).31, 40, 42 We also observed similar Dox-loading capacity of 29% and 51% at pH 4.5 and 7.4, respectively for the bt-FCBSA (Figure 5A). Whereas the Dox-loading aptitude of the BSA (Dox to BSA ratio = 1: 5 (w/w)) was measured to be 9% and 11% at pH 4.5 and 7.4, respectively (Figure 5A, S11 and Table S2). All drug-loading experiments were carried out using a fixed Dox to nanovehicle weight ratio of 1: 5.The conjugation of laurylamines to the BSA molecules allowed the nanovehicles to form hydrophobic core, which could be the driving force for the efficient encapsulation of hydrophobic Dox molecules. Such kind of hydrophobic core is absent in BSA molecules. The surface potential (ζ) measurements revealed that ζ = 8.6 ± 2 and 7.0 mV for the Dox encapsulated FCBSA (Dox@FCBSA) and free FCBSA at pH 7.4. Whereas, the ζ values were 8 ± 2 mV and 10 ± 1 mV at pH 7.4, for the Dox encapsulated bt-FCBSA (Dox@btFCBSA) and free FCBSA, respectively. The increase in diameter of the hydrodynamic sphere (dH) of Dox@FCBSA and Dox@bt-FCBSA at pH 7.4 further supports successful entrapment of Dox molecules (dH = 196 nm for Dox@FCBSA, dH = 178 nm for FCBSA, dH = 203 nm for Dox@bt-FCBSA, dH = 188 nm for FCBSA) without affecting the stability of the NPs. Figure S12 depicts the dH and morphology of the Dox@bt-FCBSA. The drug release profile of these nanovehicles is an essential tool to investigate the efficiency of the formulations with a view to understand the consequences of various formulations on the release behavior. The time-dependent release of Dox from the nanovehicles were measured in PBS buffer at pH 7.4 and 4.5 in PBS buffer (at 37 °C) by fluorescence based vesicle-leakage method.31, 40-41 The extents of Dox release from the BSA, FCBSA and bt-FCBSA were 87.12%, 69.13%, and 75% at pH 4.5 and 76.77%, 41.43%, and 49% at pH 7.4, respectively


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after 72 hours (Figure 5B). Hence, the cumulative release profiles suggest that the extent Dox released from FCBSA and bt-FCBSA at pH 4.5 is notably higher in comparison with that at pH 7.4.This burst release in acidic pH is highly beneficial to deliver Dox to the cancer cells as the microenvironment of the cancer cells is acidic in comparison with the normal cells.3, 13, 15, 19, 43 It is also important to note that the FCBSA and bt-FCBSA followed a controlled release profile, suggesting the higher stability of the Dox@FCBSA and Dox@bt-FCBSA over a longer period of time at physiological pH. However, Dox@BSA followed a burst release profile under physiological conditions. The presence of hydrophobic core inside the nanoaggregates and low positive surface potential values of FCBSA and bt-FCBSA may be the reason of its longer drug accumulation capacity.

Figure 5. Dox encapsulation and release efficiency measurement at pH 4.5 and pH 7.4 (A).Dox release profile (0-3 days) at pH 4.5 and 7.4 (B).


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This specific late onset of the extent of released Dox at physiological pH is interesting, and to the best of our knowledge this phenomenon has not been observed for other protein based nanovehicles. The burst release is usually a problem with most of the NP-based drug delivery systems.14, 38 The pH dependent Dox release profile of FCBSA and bt-FCBSA envisages their higher colloidal stability during blood circulation, sustained drug release and applicability in superior drug delivery to the cancer cells over the normal cells. Recent studies revealed that the microenvironment of the cancer cells is usually acidic in comparison with the normal cells.3, 13, 15, 19, 43

Hence, we presume that the stability and pH dependent drug release competence of FCBSA

and bt-FCBSA would have better applicability. The nanovehicles (with diameter < 400 nm) are widely used to encapsulate Dox molecules, because of its efficiency in evading the uptake by the reticuloendothelial systems. The smaller sizes also restrict the nanovehicles from renal excretion and facilitates efficient uptake due to the EPR effect for the tumor tissues in comparison with the normal tissues.2, 4, 15

Antifouling Property of the Nanovehicles ─ The presence of overall cationic surface and stability of FCBSA over a longer period of time encouraged us to investigate its interactions with protein and different types of lipids. Cellular uptake of the DDS is highly regulated by the formation of protein corona at its surface while moving through blood circulation system. Hence, resistance to protein adsorption on the surface is considered as an important characteristic property of these vehicles.23, 26, 44 We first measured this antifouling property of FCBSA and btFCBSA by monitoring the aggregation behavior of Dox@FCBSA and Dox@bt-FCBSA, respectively in 10 mM PBS at pH 7.4, containing 150 mM NaCl and 10% human blood serum for 72 hours (Figure 6A). The DLS measurements showed no significant changes in the overall


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hydrodynamic spheres of the Dox@FCBSA and Dox@bt-FCBSA, indicating its stability and non-aggregation properties in the serum containing buffer. However, the Dox@BSA showed large self-aggregation behavior, indicating predominant protein-protein interaction of the Dox@BSA with BSA under the similar experimental conditions. The antifouling properties of Dox@FCBSA and Dox@bt-FCBSA were further verified by isothermal titration calorimetry (ITC) measurements (Figure 6C).44 No measurable binding of Dox@FCBSA and Dox@btFCBSA with BSA was observed (ΔG = + 10.48 and + 2.05 Kcal/mol, respectively), suggesting its longer-time stability within the blood circulatory system (Table 1). The interaction of Dox@BSA with BSA showed a large amount of heat release (ΔG = ─ 840 Kcal/mol), indicating strong protein-protein interactions and surface adsorption (Table 1). The interaction of Dox@BSA with BSA is an enthalpy driven process, whereas the interaction of Dox@FCBSA and Dox@bt-FCBSA with BSA is entropy driven process, suggesting the release of water molecules from its binding cleft as the major thermodynamic regulatory force. Hence, the DLS and ITC measurements revealed no/negligible amount of protein binding on the surface of FCBSA, which is highly beneficial for its cellular uptake.44 However, additional experiments including proteomic analysis are required to prove the anti-fouling properties of this nanoveshicles.

Interaction of the Nanovehicles with the Model Liposomes ─ The interaction of the DDS with the membrane lipids is also considered as a key parameter for the cellular uptake properties. To investigate the interaction of FCBSA and bt-FCBSA with the different types of membranes, ITC measurements were performed using model membranes.44-45 The interaction of FCBSA, btFCBSA and BSA with zwitterionic liposome (DPPC/cholesterol (7:3)) showed almost no heat


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Figure 6. Stability of BSA, FCBSA and bt-FCBSA in the presence of human blood serum (A). ITC traces of titrating BSA (B), FCBSA (C) and bt-FCBSA (D) with BSA protein. ITC traces of titrating BSA or FCBSA with zwitterionic and anionic liposomes (E). Integrated enthalpy changes were plotted after subtracting the heat of dilution. Schematic representation that btFCBSA is resistant to the adsorption of protein and DPPC/Cholesterol membrane, allowing longer blood circulation (F).

change. But the interaction of FCBSA, bt-FCBSA and BSA with anionic liposome (DPPC/DPPS/cholesterol (4:3:3)) showed a significant amount of heat change (Figure S13-14). Particularly, the extent of heat changes for the interaction of FCBSA and bt-FCBSA with anionic liposomes was much higher, indicating its stronger interactions (Table 1). It is well documented that the outer plasma membrane of cancer and normal cells are generally anionic and zwitterionic, respectively.20 The anionic lipid, phosphatidylserine, generally constrained to the intracellular surface of the normal cells, but it gets exposed on the external surface of the most tumorigenic cell lines. Therefore, these liposome interaction measurements suggest that FCBSA or bt-FCBSA might have better uptake properties for the cancerous over the normal cell lines.

Biological Activities of the Drug Encapsulated Nanoparticles ─ The interaction pattern of protein based NPs with the serum protein and model lipids inspired us to investigate their Dox delivery efficacies in the model cancer and normal cell lines.31 The cytotoxic effect was evaluated by the MTT reduction assay in HeLa cells, which is a biotin receptor-positive model cervical cancer cell line. The outcome of the cytotoxicity assay


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Table 1. Thermodynamic parameters obtained from the isothermal titration calorimetric studies. Titrand/Analyte

Titrant/ Injectant

ΔH

ΔS

ΔG

(Kcal/mol)

(Kcal/mol/deg)

(Kcal/mol)

Dox@BSAa

BSA

-8.71×105

-2.9×103

-840.00

Dox@FCBSAa

BSA

-3372.36

-11.35

10.48

Dox@bt-FCBSAa

BSA

-1.11

-0.01

2.05

BSA

DPPC/cholesterol

NIb

-

-

FCBSA

DPPC/cholesterol

NIb

-

-

bt-FCBSA

DPPC/cholesterol

NIb

-

-

BSA

DPPC/DPPS/cholesterol

-478.00

-1.60

-1.20

FCBSA


2159.75

7.26

-2.99

bt-FCBSA


665.00

2.24

-3.27

a

Dox@BSA, Dox@FCBSA and Dox@bt-FCBSA indicates doxorubicin encapsulated BSA and doxorubicin encapsulated FCBSA, respectively. b

NI represents interaction between the analyte and the injectant was not detected.

showed that the NPs of only BSA, FCBSA and bt-FCBSA have poor toxicity for HeLa cells even up to 100 μM concentration, indicating their relevance in drug delivery applications (Figure7A and Table S3). However, the IC50 values of free Dox, Dox encapsulated BSA (Dox@BSA), Dox@FCBSA and Dox@bt‐FCBSA were 3.32, 3.83, 2.94 and 2.91 μM, respectively, in the HeLa cell line (Table S3). The MTT assay was also performed for FCBSA, bt-FCBSA, Dox@FCBSA and Dox@bt-FCBSA in HEK-293, which is a biotin receptornegative model normal cell line. The MTT results show that FCBSA and bt-FCBSA also had low toxicity up to 100 μM concentration in HEK-293 (Figure S15). These indistinguishable IC50


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values of free Dox, Dox@BSA and Dox@bt‐FCBSA also support the Dox loading efficacy of these nanovehicles.

Figure 7. Cell viability of BSA, FCBSA, bt-FCBSA, Dox, Dox@BSA, Dox@FCBSA and Dox@bt-FCBSA were measured (after 60 hours of treatment) in HeLa cells at different concentrations (A). Time-dependent Cell viability of the Dox@BSA and Dox@bt-FCBSA were


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measured in HeLa cells at the IC50 values (B). Schematic representation of the Dox loaded BSA (C) and bt-FCBSA (D) NPs that were used for cell activity studies. Flow cytometry histogram profiles of Dox@BSA and Dox@bt-FCBSA in HEK-293 (E) and HeLa (F) cells. All experiments were performed in triplicate.

The IC50 value of Dox@FCBSA and Dox@bt-FCNSA in HEK-293 cell line was 29.7 and 30.2 μM, respectively, indicating around 10-fold lower toxicity than in HeLa cell line (Table S3). The difference in IC50 values between the cell lines could be due to presence of increased number of biotin-receptors on the surface of HeLa cells. In addition, the time-dependent MTT reduction assay (at fixed concentration of Dox@BSA and Dox@bt‐FCBSA) showed that the rate of cell death was much slower for Dox@bt‐FCBSA, indicating its controlled and sustained release of Dox, which is one of the prerequisite of a successful drug delivery vehicle (Figure 7B). The outcome of time-dependent MTT assay correlates well with the Dox release profile of Dox@bt‐ FCBSA. The uptake of Dox encapsulated nanovehicles by the HeLa cells were also inspected under fluorescence and confocal laser scanning microscopes (Figure S16-17). The analysis of microscopic images of the HeLa cells suggested successful delivery of Dox by the nanovehicles. In addition, the cellular uptake of Dox by these nanovehicles were investigated by flow cytometry analysis (FL3H channel with λem = 650 nm), to eliminate the probability of fluorescence signal due to adherence of Dox-encapsulated nanovesicles to the cell surface (Figure 7E and 7F).31 The inherited fluorescence signal of Dox was used to monitor the uptake by the HeLa cells. The flow cytometry analysis also showed that Dox@bt‐FCBSA has higher uptake in HeLa cells in comparison with the HEK-293 cells. Hence, the outcome of MTT assay,


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fluorescence microscopic image analysis and flow cytometry analysis confirmed the delivery of Dox by these nanovehicles into the HeLa cells. The uptake of Dox@BSA or Dox@FCBSA would mainly depend on passive targeting as it lacks any targeting ligand on its surface. Hence its cellular internalization could proceed via different endocytic pathways. The internalization of Dox@bt‐FCBSA to the HeLa cells would proceed via active transport mechanism involving the biotin-receptors, which may help it to reduce off-target side effects of Dox.

Figure 8. Results of flow cytometry assay on HeLa cells, showing the distributions of cells present in the early and late apoptotic populations when treated with Dox, Dox@BSA and Dox@bt-FCBSA for 60 hours.


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To conform that the released Dox from the Dox@bt‐FCBSA induces apoptosis in HeLa cells, flow cytometry analysis was performed using AO/PI assay (Figure 8 and Table S4). This assay showed an increase of both early and late apoptotic cell populations, suggesting that the IC50 value of Dox@bt‐FCBSA obtained from MTT-based cell viability studies may be due to a combination of both cell cycle arrest and apoptosis. It is important to mention that the efficiency of Dox@bt‐FCBSA in inducing cell inhibitory and cell apoptosis of the HeLa cells is lower than that of free Dox. The extent of apoptosis depends on the abundance of free Dox molecules inside the nucleus of the cell. The flow cytometry assay was performed after 60 hours of the Dox treatment and the outcome of the measurement showed that the extent of cell death is lower for Dox@bt‐FCBSA in comparison with only Dox (Table S4). When cells were treated with free Dox molecules its local concentration in the nucleus is much more in comparison with that for Dox@bt-FCBSA, which could be due to its slow Dox release capability. This observation is in good agreement with the outcome of the fluorescence-based Dox release assay and timedependent MTT assay. Although, the extent of cell death after 60 hours is much higher for free Dox but, higher early apoptosis rate of Dox@bt-FCBSA could have superior advantage. The targeted delivery by bt-FCBSA nanovehicle is also advantageous because it can prolong and maintain efficient plasma concentration of the Dox molecules, minimize its cytotoxicity and maximize it site-specific delivery with controlled release propensity. To investigate the novelty of this protein-based nanovehicle (bt-FCBSA) in therapeutics, the in vivo studies were performed in an established murine Ehrlich ascites carcinoma (EAC) solid tumor model. The EAC cells were maintained in ascetic form in Swiss albino mice by intraperitoneal transplantation every 10th day on each mice. The ascetic fluid was drawn on 7th


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day on implantation according to the reported method.46 Isolated cells were diluted in sterile PBS and EAC cells (2 × 106) were then implanted in mammary fat pad of all the female mice for tumor development in breast according to the previously reported methods.47 After inoculation of the cells in mammary fat pad (on day 0) tumors were allowed to grow for 10 days followed by intravenous treatment of normal saline in group I, free Dox in group II and Dox@bt-FCBSA in group III on every alternate day for four times. All the animals were sacrificed on day 18 and tumor mass was measured post sacrifice (Figure 9A). The free Dox dose of 2.5 mg/kg was selected according to the reported methods.48 The effective concentration of Dox in Dox@btFCBSA was also 2.5 mg/kg body weight. The outcome the in vivo studies revealed that the experimental animal group treated with Dox@bt-FCBSA showed more significant tumor (breast) regression potential in comparison with that of free Dox treatment (Figure 9C-D). The reduction of tumor size was 73% and 89% in the presence of free Dox and Dox@bt-FCBSA, respectively. The body mass of the experimental animal group treated with free Dox also reduced (Table S5). Interestingly, the Dox@bt-FCBSA treated group showed less reduction of body weight, depicting its more effective therapeutic potential (Figure S18). It also can be assumed that the Dox@bt-FCBSA treatment has low toxicity in other tissues than free Dox treatment. Hence, the in vivo studies clearly demonstrate the applicability and high Dox delivery efficiency of this protein-based DDS. Ideally, these nanovehicles would be inexpensive, administrable, reduce the required dosing frequency to improve patient adherence, and minimize side effects.


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Figure 9. In vivo activities of the drug encapsulated protein-based nanovehicle (bt-FCBSA). In vivo experimental design (A). Effect of free Dox and Dox@bt-FCBSA on the progression of solid tumor in the breast of female Swiss albino mice (B-D). The breast tumor was induced in Swiss albino mice by injecting EAC cells in mammary fat pad. Four doses of free Dox and Dox@bt-FCBSA were administered to tumor bearing mice every alternate day from 10th day of EAC cell injection. Free Dox dose = 2.5 mg/kg body weight. Effective dose of Dox in Dox@btFCBSA = 2.5 mg/kg body weight (calculated using % of loading efficiency). NS = normal saline.

To investigate whether the treatments of free Dox and Dox@bt-FCBSA have any acute immunotoxicity to the mice, the immunohistochemical tests were performed. The haemotoxylin


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and eosin staining was performed to study any disruption in tissue architecture of systemic organs after the treatments of normal saline, free Dox and Dox@bt-FCBSA. Liver sections were investigated for enlargement of nuclei or Kupffer cells, ballooning of hepatocytes and centralvein congestion. The spleen and kidney tissues were examined for inflammation and alteration of basic architecture. The treatment of free Dox showed mild central vein congestion that was reduced in Dox@bt-FCBSA treated tissues and resembles as control tissues (Figure S19). A splenic contraction was observed for free Dox treated tissues. However, the splenic contraction was attenuated in Dox@FCBSA treated tissues (Figure S19). The kidney section showed intertubular haemorrhage in free Dox treated tissues which was absent in Dox@btFCBSA treated tissues (Figure S19). These finding confirms that Dox@bt-FCBSA treatment has low systemic toxicity and has better effectiveness in comparison with that of free Dox treatment. To understand whether there is any acute immune-toxicity in systemic organs after the treatments of free Dox and Dox@bt-FCBSA, we performed immunohistochemistry of proinflammatory cytokine TNF-α in liver, spleen and kidney of the experiment groups treated with normal saline, free Dox (2.5 mg/kg) and Dox@bt-FCBSA (2.5 mg/kg). Interestingly, we observed reduced expression of TNF-α in liver and spleen in free Dox treated tissues, depicting imbalanced inflammatory profile and toxicity, whereas Dox@bt-FCBSA treatment showed comparable expression of TNF-α with normal saline treatment tissues (Figure S20). However, we found no significant alteration of TNF-α expression in kidney in all the three experiment groups (Figure S20). Overall, the outcomes of immunohistochemical experiments decipher very low toxicity in systemic organs of Dox@bt-FCBSA treated tissues and better responsiveness in comparison with the free Dox treated tissues.

CONCLUSION In summary, we developed laurylamine conjugated BSA molecules, which self-assembled in aqueous solution to form cationic NPs. The MALDI-TOF mass spectrometric analysis revealed that around 50% of the total carboxylic acid groups of glutamic acid and aspartic acid residues of the BSA protein were modified with the laurylamine. The NPs were found to be highly stable


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against temperature, PH fluctuations and proteolytic enzyme stress. This nanoassembly efficiently encapsulated the model hydrophobic drug, Dox but showed its sustained release (40% after 3 days) profile. The ITC measurements showed that the FCBSA interacts very weakly with serum protein and zwitterionic liposomes, whereas it strongly interacts with anionic liposomes. The cellular uptake and cytotoxic properties were evaluated using MTT assay, fluorescence microscopy, and flow cytometry analysis, which revealed its biocompatibility and drug delivery efficiencies. An additional surface modification of these NPs with biotin derivative allowed them to successfully deliver the Dox to the biotin-receptor positive HeLa cells. The MTT assay data showed that the IC50 value of the NPs was 10-fold lower for HeLa cells than HEK-293 cells, which suggested that the biotin modification to the FCBSA played important a role in the targeting and killing of the cancer cells. The flow cytometry data analysis confirmed the targeting of biotin receptor positive cancer cells. The in vivo studies also highlighted the therapeutic potential of this protein-based nanovehicle. Therefore, the laurylamine and biotin modified BSA nanoassembly could represent a promising drug delivery system for targeting biotin receptor-overexpressing cancer cell lines, with ability to deliver hydrophobic Dox into the cells. This receptor directed nanoassembly also can be used as universal vehicle to deliver both hydrophobic and hydrophilic drug molecules to cancer tissues limiting the requirement of multiple dosages and improving patient compliance.

ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: Experimental procedures, biophysical studies, cellular and in vivo activity data (PDF).


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; Fax: +91 03 612582249; Tel: +91 03 612582217. *E-mail: [email protected]; Fax: +91 03 612582349; Tel: +91 03 612582325. ORCID Debasis Manna: 0000-0002-6920-9000 Author Contributions §

A.S. and N.P. contributed equally to this work.

Funding Sources The authors gratefully acknowledge Department of Biotechnology, Govt. of India (MED/2015/04) and Science and Engineering Research Board, Govt. of India (EMR/2016/005008) for financial support. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors are thankful to Central Instrument Facility and Department of Chemistry for instrumental support. The authors also thank Mr. Sooram Banesh of IIT Guwahati for his valuable suggestions. ABBREVIATIONS CD, Circular dichroism; CAC, critical aggregation concentration; Dox, doxorubicin; DDS, drug delivery systems; DLS, dynamic light scattering; EPR, enhanced permeability and retention;


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Fatty-Amine-Conjugated Cationic Bovine Serum Albumin

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