Detachable Polyzwitterion-Coated Ternary Nanoparticles Based on

Subscriber access provided by University of Leicester

Functional Nanostructured Materials (including low-D carbon)

Detachable polyzwitterion coated ternary nanoparticles based on peptide dendritic carbon dots for efficient drug delivery in cancer therapy Jin Ma, Ke Kang, Yujia Zhang, Qiangying Yi, and Zhongwei Gu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b17041 • Publication Date (Web): 26 Nov 2018 Downloaded from http://pubs.acs.org on November 26, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Detachable polyzwitterion coated ternary nanoparticles based on peptide dendritic carbon dots for efficient drug delivery in cancer therapy Jin Ma,†,‡ Ke Kang,† Yujia Zhang,† Qiangying Yi, *,† Zhongwei Gu*,†, § † National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu 610064, P.R. China; ‡ Department of Cell and Chemical Biology, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands. § College of Materials Science and Engineering, Nanjing Tech University, 30 South Puzhu Road, Nanjing 211816, P. R. China. KEYWORDS: Zwitterion, Peptide dendrimer, Carbon dots, Drug delivery, Cancer Therapy

ABSTRACT

In this work, we presented the ternary nanoparticles [pCBMA(CD-D/DOX)] based on peptide dendritic carbon dots (CDs) to realize tumor-specific drug delivery and high efficient cancer therapy. The versatile nanoparticles could achieve “stealth” delivery in blood due to the anti-fouling zwitterion coating. Meanwhile, charge 1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 50

changes of the zwitterions could be moderated during their transportation toward/inside tumor cells, where subtle environmental pH variations acted as potent stimuli to actualize desired functions. In particular, the detachment of the zwitterionic “coat” at tumor site resulted in exposure of the abundant peripheral guanidine groups on peptide dendritic carbon dots (CD-D/DOX) owing to the extracellular pH environment (pH 6.8) induced charge conversion. Consequently, the positively charged CD-D/DOX (+7.02 mV) interacted with the negatively charged cancer cell membrane to enhance cellular uptake. After endocytosis, tumor intracellular microenvironments (acidic condition and high glutathione level) could launch effective disintegration of the CD-D/DOX entities due to acid-induced protonation of guanidine groups and glutathione-induced cleavage of peptide dendritic components on CDs, and then effective endosomal escape and fast doxorubicin hydrochloride (DOX·HCl) release (73.2 % accumulative release within 4 h) were achieved successively. This strategy enabled a 9.19-fold drug release rate at tumor sites in comparison with the one at the physiological environment. Moreover, the excellent fluorescent property of CDs endowed the pCBMA(CD-D/DOX) fluorescence bioimaging function. In view of the above-mentioned

advantages,

pCBMA(CD-D/DOX)

exhibited

outstanding

anti-tumor activities both in vitro and in vivo, demonstrating much higher anti-tumor efficacy and less side effects than the free DOX·HCl.

INTRODUCTION 2 ACS Paragon Plus Environment

Page 3 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Malignant tumors have been the main cause of death worldwide and chemotherapy is one of the most commonly used approaches in clinical treatments.1-2 However, due to the unspecific delivery and low bioavailability, antitumor drugs in free formulation generally cause serious side effects on healthy tissue and decreased therapeutic efficacy.3-4 Encouragingly, nanoscale drug delivery systems (DDSs) have been developed as promising platforms to enhance bioavailability of antitumor drugs and thereby improve therapeutic index and reduce the systemic toxic effect owing to their high efficient delivery of therapeutic cargos to tumor sites. Typical examples of the DDSs have been demonstrated as the liposomes, micelles, polymers, dendrimers, organic or inorganic nanoparticles.5-7 Very recently, theranostic nanomaterials which combine the chromophores and antitumor drugs have been designed to achieve synchronous fluorescence bioimaging and drug delivery.8-9 Nevertheless, conventionally used fluorescent organic dyes or hosts limited their theranostic applications in clinical for several drawbacks, for example the fluorescence quenching, poor water solubility, poor biocompatibility, nonbiodegradation, etc.10 On the contrary, fluorescent CDs with remarkable superiority including excellent biocompatibility, water solubility, excitation-dependent emission tenability, low toxicity and rich in modifiable surface functionalization, have been developed as excellent fluorescent probes for bioimaging.11-14 Moreover, it has been reported that antitumor drug DOX·HCl bearing anthracene rings and amino groups could non-covalently attach to CDs by electrostatic interactions and π–π stacking, 3 ACS Paragon Plus Environment


Page 4 of 50

which were sensitive to tumor microenviromental pH conditions, therefore cancer-specific drug release could therefore be realized.15-16 Thus, development of the therapeutic nanoplatforms utilizing CDs would greatly simplify the loading procedure of the anthracycline drugs, largely enhance corresponding loading capability and integrate preferred fluorescence property to satisfy the clinical requirements. The DDSs have shown great capability to increase solubility and stability of the loaded therapeutic agents. However, during their transportation in circulation, the cells and abundant plasma proteins in blood could nonspecifically adsorb onto the DDSs and consequently lead to their elimination by the mononuclear phagocyte system (MPS).15 On this basis, the most essential work for developing of multifunctional nanocarrier systems should focus on how to avoid nonspecific adhesion of environmental biomolecules to nanocarriers, in order to reduce large accumulation of the anti-cancer drugs at healthy metabolic organs and improve the

drug

bioavailability.

Numerous

non-fouling

materials/coatings

(e.g.,

polysaccharides, ethylene glycol-based polymers and oligomers) that are usually well hydrated with neutral or weakly negative charges have therefore been employed to achieve the goal.18 In particular, the zwitterions, which possess super-low fouling properties owing to their hydrophilia and low interfacial energy, have been demonstrated as great anti-fouling candidates in DDSs.19 As reported in our previous work, the zwitterionic decoration [poly(carboxybetaine methacrylate), pCBMA] outside the micelles considerably reduced the nonspecific 4 ACS Paragon Plus Environment

Page 5 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


protein adsorption and achieved “stealth” delivery to prolong the circulation time of functionalized micelles in blood.20 Beyond that, advantage of the zwitterions has also been strongly supported by their pH-liable charge variability. It has also been revealed in our work previously, surface charge of the built zwitterionic micelles could reverse from negative (-2.4 mV) in the blood where the pH was around 7.4 to positive (+4.41 mV) after targeting to tumor sites where the pH was slightly decreased to 6.8.20 Thus, application of these zwitterions for surface functionalization would generate a facile way to control over the surface charge of nanoscale drug carriers at varying pH conditions, where desired functions of the nanocarriers could be easily accomplished. Another major challenge for successful drug delivery and subsequent tumor cell killing is the required efficient tumor cellular internalization and endosomal escape after enrichment of the nanocarriers at tumor sites.21 It has been proven that conversion of the surface charge of the nanocarriers from negative to slightly positive after blood circulation could facilitate the cellular uptake via electrostatic interaction-mediated internalization; on the other hand, the “Proton Sponge” effect at relative acidic cellular compartments (e.g., endo-lysosomes, pH 4.0-5.5) could lead to successful endosomal escape of the active therapeutic substances before one could work at full capacity. For this purpose, functional building blocks in a smart DDS should be able to actualize above expected transitions at given tumor microenvironments to meet all requirements simultaneously. Hopefully, amphiphilic dendrimers with precisely controlled “tree”-like architecture and 5 ACS Paragon Plus Environment


Page 6 of 50

amplified functions could be recommended to cope with this challenge.22 As a typical classical drug delivery vehicles, peptide dendrimer-based nanocarriers have been widely studied by Gu’s group, owing to their abundant peripheral functional groups (e.g., 16 active groups for the three-generation peptide dendrimer)23-24 and biodegradable chemical composition.25 In our work, to ensure safe transportation of nanocarriers into/inside tumor cells, the peptide dendrimer derived from arginine would be the powerful alternative due to its terminal guanidine which can interact with pCBMA at neutral conditions and be ionized to detach pCBMA and promote drug endosomal escape. Herein, a tumor specific multiple responsive zwitterionic nanoplatform composed of dendrimer modified CDs together with polyzwitterion (pCBMA) decoration has been developed for cancer therapy (Scheme 1). Strategically, the versatile nanoparticles could achieve “stealth” delivery in blood due to the anti-protein pCBMA zwitterions and could take off the zwitterionic “coat” at mild acidic tumor microenvironment (~ pH 6.8) and disintegrate into smaller sub-nanoparticles (arginine dendrimer decorated CDs with DOX loading) after enrichment at tumor sites. Then, exposure of the abundant peripheral guanidine groups in the arginine dendrimers would endow the sub-nanoparticles positive charge which makes important sense in enhancing the interaction with negatively charged cell members and further facilitates endocytosis of the sub-nanoparticles. Moreover, acidic intracellular compartments would protonate guanidine groups in the peptide dendrimers to help drug endosomal escape effectively. Last but not least, 6 ACS Paragon Plus Environment

Page 7 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the acid (pH 4.5-5.5) and highly reductive (~5 mM glutathione) conditions inside tumor cells could desorb DOX from CDs and cleave the disulfide linkages (S-S) between the dendrimers and CDs to speed up nucleus-specific drug release for tumor treatment. EXPERIMENTAL SECTION Materials. Glucose and poly(ethylene glycol)-200 (PEG200) were purchased from Kelong Chemicals (Chengdu, China). L-Lys-OMe·2HCl and Boc-Arg(Pbf)-OH were purchased

from

GL

Biochem

(Shanghai,

China)

Ltd.

1-ethyl-3-[3-dimethylaminopropyl]

carbodiimidehydrochloride

(EDC),

N-Hydroxysuccinimide

N,N-Diisopropylethylamine

(DIEA),

(NHS),

1-hydroxybenzotriazole

(HOBt),

N,N,N’,N’-tetramethyl-O-(1H-benzotriazol-1-yl)uronium (HBTU),

trifluoroacetic

acid

1,1,4,7,10,10-Hexamethyltriethylenetetramine

(TFA),

hexafluorophosphate CuBr

(HMTETA,

(99.99%), 97%),

ethyl

α-bromoisobutyrate (EBiB, 98%), cystamine dihydrochloride, dithiothreitol (DTT), methanol (MeOH), N,N-Dimethylformamide (DMF) and other solvents were purchased from Sigma-Aldrich. β-propiolactone (95%) was purchased from Aladdin (China). Doxorubicin hydrochloride (DOX·HCl) was purchased from Hisun Pharmaceutical (Zhejiang, China). Cell counter kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). Penicillin, streptomycin and fetal bovine serum (FBS) were purchased from Hyclone (USA). 4T1 cell line (mouse breast 7 ACS Paragon Plus Environment


Page 8 of 50

cancer cell), HepG2 cell line (Human hepatoma carcinoma cell) were purchased from Chinese Academy of Science Cell Bank for Type Culture Collection (Shanghai, China). BALB/c mice (14 ± 2 g, 4-5 weeks old) were purchased from Dashuo Experimental Animal Company (Sichuan, China). Methods. 1H-NMR spectra were obtained by using 400 MHz Bruker Advanced Spectrometer. The molecular weight was measured by matrix-assisted laser desorption ionization time-of-light mass spectrometry (MALDI-TOF-MS, Bruker, USA). The size and zeta potential were determined by a Malvern Zeta-sizer Nano ZS at 25 °C. The morphology was observed by Scanning electron microscopy (SEM, S-4800

Hitachi),

transmission

electron

microscope

(HRTEM,

FEI

Tecnai

GF20S-TWIN, USA) and atomic force microscopy (AFM, MFP-3D, Asylum Research, USA). The fluorescence properties were studied by a fluorescence spectrophotometer (Hitachi F-7000, Japan). Ultraviolet–visible absorbance spectra were recorded by a UV-Vis spectrophotometer (PerkinElmer Lambda 650S). Fourier transform infrared (FTIR) spectra were obtained by a FT-IR PE spectrometer using the KBr pellet technique with a wave number in the range of 4000-500 cm-1. Preparation of CDs. Glucose (2 g) was dissolved into 6 mL distilled water in a 100 mL beaker and then 20 mL PEG 200 was added under stirring.26 After forming a transparent solution, the beaker was placed into a microwave oven and heated at 800 W for 3 min. The color of the solution was turning from transparent to dark brown which implied the formation of CDs. After cooling to room temperature, the CDs 8 ACS Paragon Plus Environment

Page 9 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


were dialyzed against water in a dialysis bag (MWCO = 3500) overnight in dark and filtered through a 0.45-μm membrane filter. Finally, the CDs suspension was diluted to 100.00 mL with water and stored at 4 ˚C for further use. Preparation of peptide dendrimer (D-S-S-D). H-Lys-OMe·2HCl (5.00 g, 21.51 mmol), Boc-Arg(Pbf)-OH (27.18 g, 51.61 mmol), EDC·HCl (9.89 g, 51.61 mmol) and HOBT (6.97 g, 51.61 mmol) were dissolved in anhydrous dichloromethane (DCM, 30 mL) under nitrogen atmosphere. After stirred in ice-bath for 30 min, DIPEA (9.02 mL, 54.56 mmol) was added dropwise. Subsequently, the solution was stirred at room temperature for 24 h. The solvent was removed by rotary evaporation and dissolved in 60 mL chloroform (CHCl3). Next, the solution was washed with HCl (1M), saturated NaHCO3 and NaCl solutions several times, respectively. The organic phase was dried with anhydrous MgSO4 for 12 h and the filtrate was concentrated by rotary evaporation, and compound 1 (Figure S1) was obtained. MALDI-TOF-MS: (m/z, [M+H]+): 1178 (observed). Then, NaOH/MeOH (1 M) was added to deprotect the methoxy group. After removal of the MeOH, CHCl3 was added to dissolve the residues. HCl (1 M) was added to adjust the pH of the aqueous phase. The organic phase was dried by anhydrous MgSO4, filtered, and concentrated to obtain the compound 2 (Figure S1) as white powder. MALDI-TOF-MS: (m/z, [M+H]+): 1164 (observed). Compound 2 (0.45 g, 0.39 mmol), cystamine dihydrochloride (28.88 g, 0.19 mmol), HBTU (0.18 g, 0.45 mmol) and HOBt (0.07 g, 0.45 mmol) were dissolved in anhydrous dichloromethane (DCM, 30 mL) under nitrogen atmosphere. After stirring in an ice-bath for 30 min, DIEA (0.23mL, 1.35 mmol) was added 9 ACS Paragon Plus Environment


Page 10 of 50

dropwise. Subsequently, the solution was stirred at room temperature for 24 h. Then the solution was evaporated and replaced with 60 mL CHCl3. After washing with HCl (1M), saturated NaHCO3 and NaCl solutions for several times, the organic phase was dried with anhydrous MgSO4 for 12 h. The compound 3 (Figure S1) was obtained after solvent evaporation. MALDI-TOF-MS: (m/z, [M+H]+): 2444 (observed). Compound 3 was treated with TFA (10 equiv, according to the number of Boc and Pbf groups) to remove protective groups in dry CH2Cl2. After stirring for 10 h at room temperature, the solution was concentrated and treated with anhydrous diethyl ether. White solid product (compound 4, Figure S1) was obtained.

MALDI-TOF-MS:

(m/z, [M+H]+): 1034 (observed). Synthesis of peptide dendrimer modified carbon dots (CD-D). The synthetic route was demonstrated in Figure S2. First, the thiolated CDs (CD-SH) were prepared. In brief, EDC·HCl (1.31 g, 6.84 mmol) and NHS (0.78 g, 6.84 mmol) were added into 80 mL CDs suspension and the mixture was stirred for 30 min in dark. Subsequently, cystamine dihydrochloride (0.52 g, 3.42 mmol) was dissolved into the solution and the reaction was carried out overnight. After purification by dialysis against water for 24 h, DTT (3.52 g, 22.86 mmol) was added to the mixture to cleave the disulfide (-S-S-) linkage and further dialysis against water for 48 h (MWCO = 1000). The dialysate was collected and lyophilized to obtain the brown solid for further study. Then, the prepared CD-SH (0.10 g) and compound 4 (0.16 g, 0.15 mmol) were dissolved in a borate buffer solution (0.01 M, pH = 8) and stirred for 4 h at room temperature. The crude product was purified by dialysis against water for 48 h 10 ACS Paragon Plus Environment

Page 11 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


((MWCO = 1000), followed by filtration through a membrane filter (0.45 µm). Then the CD-D was obtained after freeze-drying. Synthesis of poly(carboxybetaine methacrylate) (pCBMA). pCBMA was synthesized according to our previously reported method (Figure S2)20 through the atom-transfer radical polymerization (ATRP). Generally, the CBMA, EBiB and HMTETA were used as monomer, initiator and ligand, respectively.

They were

placed in a flask under a Schlenk condition and dissolved in MeOH/DMF (1:1) under protection of nitrogen. After addition of the catalyst CuBr, the flask was transferred to an oil bath at 60 °C. The resulting solution was stirred for 24 h, followed by dialysis against water for purification (MWCO = 1000). Finally, the pCBMA was lyophilized for further use. Preparation and characterization of pCBMA(CD-D/DOX). CD-D (20 mg) and DOX·HCl (5 mg) were dissolved in ultrapure water with stirring for 4 h. After the DOX·HCl was fully adsorbed on the surface of CD-D, the mixture was dialyzed against water for 24 h to remove excess DOX·HCl. Finally, the solution was lyophilized to obtain the sub-nanoparticles. The sub-nanoparticles (10 mg) and pCBMA (5 mg) were dissolved in ultrapure water with stirring for overnight. Then the mixture was filtered through a membrane filter (0.45 µm), followed by freeze-drying. DOX loading efficiency and content were studied by a UV-Vis spectrophotometer.

11 ACS Paragon Plus Environment


Page 12 of 50

Drug loading efficiency (DLE) and drug loading content (DLC) were calculated according to the following formula: DLC (%) = W/W0 × 100%

(1)

where the W was the weight of DOX in pCBMA(CD-D/DOX), W0 was the weight of DOX·HCl initially added in drug loading procedure. DLC (%) = WDOX / WTotal × 100%

(2)

where the WDOX was the weight of DOX in pCBMA(CD-D/DOX), WTotal was the weight of pCBMA(CD-D/DOX). in vitro drug release. The pCBMA(CD-D/DOX) was accurately weighed and re-dispersed in PBS (0.01 M, pH = 7.4) or PBS (0.01 M, pH = 5.5,) containing DTT (10 mM). The suspensions were transferred to the dialysis bags (MWCO = 3000-5000) and immersed in large volume centrifuge tubes containing 20 mL of PBS with varied pH and redox conditions (pH 7.4; pH 5.5, 10 Mm DTT). The samples were incubated at 37 °C under mild shaking for responsive release. At given time, 1 mL of dialyzate was taken for analysis followed by replenishment of fresh buffer solution with the same volume immediately. The concentration of released DOX in dialyzate was detected using a UV-Vis spectrophotometer by measuring the absorbance at 480 nm, and the quantized data was used to describe the triggering-responsive drug release profiles. All the measurements were carried out in triplicate.


Page 13 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Protein adsorption. We used bovine serum albumin (BSA, 66 kDa) to investigate possible

protein

adsorption

on

the

pCBMA(CD-D/DOX).

BSA

and

pCBMA(CD-D/DOX) was dissolved into PBS together to reach 0.30 mg/mL and 0.15 mg/mL separately. After incubation for certain time (0.5 h, 1 h, 2 h, 4 h) at 37 °C under shaking, the mixture was centrifuged at 13000 g for 15 min to precipitate the protein that adsorbed onto the pCBMA(CD-D/DOX). BCA Protein Assay Kit (BD, Rockford IL, USA) was used to detect the amount of unabsorbed protein. Absorbance was measured at 562 nm with a microplate reader (Thermo Scientific, VARIOSKAN FLASH, USA). Confocal laser scanning microscopy. The 4T1 cells (1 × 104 cells per well) were seeded in 35 mm glass-bottomed dishes and allowed to adhere for 24 h. After removing the culture medium, the pCBMA(CD-D/DOX) were diluted in culture medium to reach a final DOX concentration of 10 μg/mL. After incubation for 2 h, 6 h, and 16 h at 37 °C, the culture medium was carefully removed. The cells were washed three times with PBS and the culture medium eventually was replaced with 300 μL PBS. Then, the cells were observed using confocal laser scanning microscopy. CDs and DOX were excited by 405 nm and 488 nm wavelength laser, respectively. For endosomal escape study, after incubation with the pCBMA(CD-D/DOX) for 0.5 h, 2 h, and 4 h at 37 °C, the culture medium was carefully removed and the acidic compartments (i.e., endo-lysosomes) were stained with LysoTracker® green DND-26 (ThermoFisher Scientific) for 30 min. The cells were washed three times with PBS 13 ACS Paragon Plus Environment


Page 14 of 50

and the culture medium was eventually replaced with 300 μL PBS. Then, the cells were observed using confocal laser scanning microscopy (CLSM, Leica TCP SP5). Cellular internalization of the pCBMA(CD-D/DOX). For flow cytometry tests, 4T1 cells (2 × 105 per well) were seeded in 6-well plates and allowed to adhere for 24 h. Fresh culture medium with the pCBMA(CD-D/DOX) (DOX concentration = 2.5 μg/mL) were added. After incubation for 2 h and 16 h at 37 °C, culture medium was carefully removed and followed by thrice washed with cold PBS. Then the cells were collected and resuspended in 0.3 mL PBS buffer after twice centrifugation at 1000 rpm for 5 min. Flow cytometry (BectoDickinson, USA) was consequent applied to detect the pCBMA(CD-D/DOX) uptake behavior by 4T1 cells (λex = 480 nm, λem = 590 nm). Each test was repeated in triplicate. in vitro antitumor therapeutic efficacy. CCK-8 (cell counting kit-8) assay was carried out to test the cytotoxicity of pCBMA(CD-D/DOX). HepG2 cells or 4T1 cells were seeded in 96-well plates (5×103 cells per well) and allowed to adhere for 24 h. CDs, D-S-S-D, DOX·HCl, pCBMA and pCBMA(CD-D/DOX) were diluted to different concentrations by fresh medium and then replaced the old medium. After exposing the cells to the compositions for 24 h and 48 h, the culture medium was removed and the cells were carefully rinsed with PBS (0.01M, pH 7.4) for three times. Serum free medium containing CCK-8 was added and incubated for 2 h at 37 °C. The absorbance at 450 nm of each well was measured to calculate cell viability according to the control group that without treatment. 14 ACS Paragon Plus Environment

Page 15 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Biodistribution. Biodistribution of pCBMA(CD-D/DOX) in mice was evaluated via monitoring the DOX accumulation in tissues/organs over time by the ex vivo fluorescence imaging. pCBMA(CD-D/DOX) re-dispersed in 200 μL saline was injected intravenously to the tumor (~150 mm3) bearing BALB/c mice through the tail vein at a DOX dose of 3 mg/kg. At the time of 6 h, 24h, and 48h, the mice were sacrificed, the tumor and major organs (heart, liver, spleen, lung and kidney) were excised. The emission fluorescence of the tissues was collected by using the 450 nm excitation filter. in vivo pharmacokinetic study. BALB/c mice (no gender preference) were treated with DOX or pCBMA(CD-D/DOX) via the tail vein at a concentration of 3 mg DOX per kg body weight. At designed time point (3 min, 30 min, 1 h, 2h, 6 h, 12 h) after administration, blood samples were collected. After centrifugation at 3000 g for 3 min, 100 µL aliquot of plasma was taken and incubated with 0.5 mL of chloroform/isopropanol (4:1, v/v) for 2 min. Then the mixture was separated by centrifuging at 10000 g for 5 min. The organic phase was collected and dried under nitrogen atmosphere. After dissolved the residue into 1 mL DMSO, the samples were centrifugation at 10,000 g for 3 min and the supernatant was tested by fluorometer. The standard curve of DOX in plasmid was established by a serially diluted DOX in plasma. Pharmacokinetic parameters were analyzed from the average DOX concentration in blood at varied times.



Page 16 of 50

in vivo antitumor efficacy. 4T1 cells (5 × 105) were utilized to establish tumor models by subcutaneous injection to female BALB/c mice. After tumors reaching about 100 mm3, the mice were randomly divided into 3 groups (n = 5) and treated with

200

μL

of

three

different

formulations

(Saline,

DOX·HCl

and

pCBMA(CD-D/DOX)) through tail veins at a dose of 3 mg DOX per kg body weight at certain time intervals. Tumor length (L) and width (W) were measured by a vernier caliper before each injection and used to calculate the tumor volume (V) according to the formula V = LW2/2. The body weight of each mouse was carefully recorded at the same time. Histological and immunohistochemical analyses. After 15 days treatment, all the mice were euthanized. Tumors and major organs (liver, heart, spleen, lung and kidney) were isolated and fixed with 4% formalin. Then the organs were used to evaluate histopathological by Hematoxylin and eosin (H&E) staining. Besides, angiogenesis, tumor cell proliferation activity and tumor apoptosis level were detected by using platelet/endothelial cell adhesion molecule-1 (CD31), monoclonal antibody Ki-67, terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) stain separately. RESULTS AND DISCUSSION Preparation and characterization of CDs. CDs were prepared through microwave pyrolysis approach with glucose as carbon source and poly(ethylene glycol) (PEG-200) as surface passivation agent.27 After purification, the CDs was around 3 16 ACS Paragon Plus Environment

Page 17 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


nm in diameter (Figure 1a), and their suspension was obtained as light brown transparent solution (Figure 1a, insert). The morphology of the CDs was confirmed by TEM measurements, suggesting uniformly dispersed spherical particles (Figure 1b). The particle size calculated from TEM images was in good accordance with the DLS results. Further magnified HRTEM images showed the 0.208 nm well-resolved lattice fringes which were close to the (020) diffraction plane of sp2 graphitic carbon (Figure 1c).15 Moreover, FTIR spectrum revealed the presence of multiple functional groups on the surface of CDs (Figure 1d). The peaks at 1644 cm-1 and 3373 cm-1 were assigned to the stretching vibration of C=O and –OH respectively, suggesting the existence of –COOH and –OH groups on the CDs surface. The vibrational absorption band of C-O-C and C-H were also detected at about 1100 cm-1 and 2943 cm-1. The carboxyl group on the CDs surface formed the basis of subsequent surface functionalization (and will be discussed below). Photoluminescence is one of the most glamorous characteristics of CDs.28 To explore the optical properties of the CDs, UV-vis absorption and photoluminescence (PL) spectroscopy were studied. As illustrated in Figure S3, the absorption peak around 303 nm in the UV-vis spectrum of the CDs was ascribed to the n-π* transition of C=O group. Moreover, the PL spectra demonstrated an excitation wavelength-dependent photoluminescent behavior of the CDs (Figure 1e). Increasing the excitation wavelength from 320 to 500 nm red-shifted the maximum emission peaks from 440 to 550 nm. When the excitation wavelength was 360 nm, the CDs exhibited the strongest emission intensity. This special excitation-dependent emission phenomenon which 17 ACS Paragon Plus Environment


Page 18 of 50

resulted from different energy levels was associated with various surface states formed by distinct functional groups on the surface of CDs. In addition, under a fluorescent microscope, a drop of the CD suspension exhibited blue, yellow and red photoluminescence under ultraviolet (330–385 nm), blue (450–480 nm) and green (510–550 nm) light excitation respectively (Figure S4). The photoluminescence property uncovered that the CDs had potential to be used as biological imaging agents. Preparation and characterization of peptide dendrimers. The synthesis route for the peptide dendrimers was shown in Figure S1. In brief, the protected generation one peptide dendrimer (compound 1) was synthesized through the condensation reaction of Boc-Arg(Pbf)-OH and L-Lys-OMe·2HCl. The compound 1 was treated with NaOH/MeOH (1 mol/L) to deprotect the methoxy group to obtain the compound 2. Then cystamine was introduced to link two compound 2 molecules together to get compound 3. Finally, after trifluoroacetic acid (TFA) treatment, the protecting groups (Boc and Pbf) were deprotected and the final product compound 4 (D-S-S-D) was obtained. All the products were characterized by 1H-NMR spectra and matrix-assisted laser desorption ionization time-off light mass spectroscopy (MALDI-TOF-MS) analysis and the data were presented in Figure S5. The MALDI-TOF-MS result of compound 4 was well agreed with the theoretical range of molecular weight distribution.


Page 19 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Preparation and characterization of peptide dendrimer modified carbon dots (CD-D). Zeta potential of the CDs was -11.33 mV (Figure 2a), further confirming the presence of numerous carboxyl groups on the surface of CDs. The existence of abundant carboxyl groups would not only improve the dispersibility and stability of the CDs in aqueous systems, but also offer various opportunities for further surface modifications. In this work, the peptide dendrimers were conjugated onto the CDs (Figure S6). Firstly, sulfhydryl groups were introduced into the CDs. As shown in Figure S7, the 1H-NMR spectrum of CDs only had two signals centered at 3.64 ppm and 3.57 ppm. Compared with CDs, two emerging absorption peaks at 3.30 ppm and 2.30 ppm in the 1H-NMR spectrum of thiolated CDs (CD-SH) were assigned to the methylene protons of the cysteamine moieties. Additionally, the FTIR was also carried out to verify the structure of CD-SH. As shown in Figure S8, unlike that of the parent CDs, the stretching vibration band of N-H at 3297 cm-1, and the stretching vibration band of C=O at 1733 cm-1 in the FTIR spectrum of CD-SH indicated the successful modification of cysteamine on CDs. Moreover, CD-SH was negatively charged with a zeta potential of -10.82 mV, which was lower than that of CDs (-11.33 mV), indicating that partial carboxyl groups were substituted with cysteamine (Figure 2a). After that, disulfide exchange reaction was used to prepare CD-D. As evidenced by the apparent contrast difference in the 1H-NMR spectra of CD-D and CD-SH, the peptide dendrimer was successfully modified onto CDs (Figure S7). Similarly, new bonds at 1663 cm-1, 1559 cm-1, 1115 cm-1 corresponded to the stretching vibrations of C=N, N-H, C-N respectively, which indicated the formation of CD-D covalent 19 ACS Paragon Plus Environment


Page 20 of 50

complexes (Figure S8). Furthermore, due to the presence of guanidine groups in the peptide dendrimer modified CDs, the net negative surface charge was drastically reduced to be -0.80 mV (Figure 2a). Preparation

and

characterization

of

Poly(carboxybetaine

methacrylate)

(pCBMA). The CBMA was synthesized according to our previous work.20 1H-NMR was used to verify the successful polymerization of the CBMA monomers. As shown in Figure S9 a and b, disappearance of the chemical shifts at 5.6 and 6.0 ppm representing vinyl groups of the CBMA monomers indicated the successful synthesis of pCBMA. To evaluate the molecular weights distribution of pCBMA, GPC and MALDI-TOF-MS detection were carried out. As the shown in MALDI-TOF-MS result (Figure S9 c), the most abundant peak of molecular weight was around 4.7 kD. In addition, coexistence of tertiary amine and carboxyl groups in pCBMA made this zwitterionic polymer possible to show a pH-dependent charge fluctuation. To elucidate the variation tendency, the effects of pH on the charge fluctuation of pCBMA were investigated. As shown in Figure 2b, the surface charge of pCBMA was -1.62 mV in pH 7.4 phosphate buffer solution due to partial ionization of carboxyl groups. When the environmental pH reduced to 6.8, suppression of the carboxyl ionization and encouragement of tertiary amine protonation led to an overall positive charge (+7.65 mV). This trend persisted up to pH 5.5, the charge of pCBMA became +13.37 mV. The pH-induced charge reversal of this zwitterionic polymer made them have great potential to be applied in constructing charge-convertible nanoparticles. 20 ACS Paragon Plus Environment

Page 21 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Preparation and characterization of nanoparticles [pCBMA(CD-D/DOX)]. Anticancer drug DOX·HCl that has a pharmacodynamic effect on cell nuclei via damaging the DNA structures was selected as a model drug to fabricate the nanosized pCBMA(CD-D/DOX). Because of the amine (–NH2) moiety and anthracene in DOX and the carboxyl group (–COOH) on the CD-D surface, the selected drug DOX·HCl could adsorbed onto CD-D surface via electrostatic interaction and π-π stacking to form a conjugation (CD-D/DOX). As shown in Figure S3, the UV-vis spectrum of CD-D/DOX presented two absorption peaks centered around 480 nm and 303 nm, which were corresponded to the absorbance of DOX·HCl and CD respectively. This result was unambiguous evidence demonstrating the successful binding of DOX·HCl and CD-D. The FTIR spectra (Figure S8) also confirmed the generation of CD-D/DOX. The new peaks at 1582 cm-1 and 1617 cm-1 were assigned to the stretching vibration of CH groups in the pyranoid ring of DOX. In addition, the zeta potential of CD-D/DOX which was completely converted to positive (+7.83 mV) after DOX conjugation further confirmed the successful construction of the non-covalent complex (Figure 2a). Size distribution of the CD-D/DOX was also investigated by DLS (Figure S10). The size of CD-D/DOX was mildly increased to 5.8 ± 0.8 nm but the CD-D/DOX still exhibited as small particles as compared to the parent CDs (~3 nm). At pH 7.4, the negatively charged linear polymer pCBMA (-1.62 mV) worked with the positively charged CD-D/DOX (+7.83 mV) to form nanoparticles via electrostatic interaction. Specifically, when the mass ratio of pCBMA and CD-D/DOX was 1:2, 21 ACS Paragon Plus Environment


Page 22 of 50

they self-assembled into well-defined nanostructures with the size distribution of 183 ± 27 nm, as shown in DLS result (Figure 2c). Scanning electron microscope (SEM) and atomic force microscopy (AFM) were employed to analyze the morphology of pCBMA(CD-D/DOX). In the SEM image (Figure 2d), pCBMA(CD-D/DOX) appeared spherical and were well dispersed. The AFM three-dimensional (3D) architectures and section analysis (Figure 2e, f and g) also revealed that the pCBMA(CD-D/DOX) were spherical nanoparticle and possessed an average diameter of approximately 200 nm. The amount of DOX loaded in the pCBMA(CD-D/DOX) was quantitatively assayed using UV-vis spectroscopy. According to the standard calibration curves obtained from DOX·HCl solutions with known concentrations (Figure S11), the drug loading efficiency (DLE) and drug loading content (DLC) were calculated to be 96.9% and 16.1%, respectively. The extraordinarily high loading capacity, which was much higher than those of the most reported cases,29-30 was attributed to the numerous carboxyl groups in the CD and the presence of electrostatic interaction and π-π stacking between CD-D and DOX·HCl. Under mild acidic tumor extracellular microenvironment (pH 6.8), the negative charge of pCBMA could be changed to positive (+7.65 mV) due to the tertiary amine protonation, and the positively charged CD-D/DOX would consequently repel the positively charged polymer. This property was confirmed by the obvious pH-dependent

zeta

potential

increase

of

the

pCBMA(CD-D/DOX)

under 22

ACS Paragon Plus Environment

Page 23 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


physiological and tumor environments. At physiological condition (pH 7.4), the pCBMA(CD-D/DOX) were stable as their hydrodynamic diameter showed no obvious fluctuation and the zeta potential was near electric neutral within 48 h (Figure 3a, Figure S12). Decrease of environmental pH (pH 6.8) led to increase of the charge to +7.02 mV, which was attributed to the charge conversion of pCBMA and accompanied exposure of positive CD-D/DOX after electrostatic repulsion. After taking off the zwitterionic “coat”, the pCBMA(CD-D/DOX) became strongly positive at pH 5.5 (+14.6 mV), indicating their further disintegration. This zeta potential situation did not change obviously even when the incubation time was prolonged to 4 h. Therefore, it was suggested that these pCBMA(CD-D/DOX) would stay stable in circulation in bloodstream (pH 7.4), and be disassembled hierarchically after collected at tumor sites (e.g., pH 5.5). Moreover, the release behavior of DOX from the assemblies was investigated in various biomimetic conditions. First, three portions of identical pCBMA(CD-D/DOX) samples were immersed in 1 mL acidic buffers solutions (pH 7.4, 6.8, 5.5) for 4 h and then the fluorescence emission spectrum at the excitation wavelength of 480 nm was examined to analyze the amount of released DOX in the buffers with different acidic strength. As shown in Figure S13, corresponding change of fluorescence intensity displayed a pH-dependent manner. Generally, the fluorescence intensity increased significantly with decreasing of environmental pH, demonstrating more fluorescent DOX molecules released at low pH condition. Furthermore, in vitro DOX release profiles of pCBMA(CD-D/DOX) were investigated in pH 7.4 or pH 5.5+10 mM DTT 23 ACS Paragon Plus Environment


Page 24 of 50

buffers. It was worth noting that the release rate and accumulative amount of DOX at pH 5.5+10 mM DTT condition was much higher than that at pH 7.4 (Figure 3b). After 1 hour, 24.9% of DOX was released into the reservoir at pH 5.5+10 mM DTT buffer, while only 3.9% of DOX was released into pH 7.4 buffer. After 4 h, a larger amount of DOX (73.2%) was released at pH 5.5+10 mM DTT condition obviously, comparing with 8.7% at pH 7.4. At 24 h, almost 9-fold released drug (78.1%) was detected in pH 5.5+10 mM DTT buffer compared with a very low value of total release (8.5%) at pH 7.4, indicating that the pCBMA(CD-D/DOX) behaved a highly selective release at acidic tumors sites and negligible DOX release at the neutral condition. To further verify that the pCBMA(CD-D/DOX) could fast disassemble and release the loaded DOX at pH 5.5+10 mM DTT condition primarily but remain stable at pH 7.4, we observed its morphology by TEM. As shown in Figure 3 c and d, after exposure to pH 5.5+10 mM DTT for 4 h, the pCBMA(CD-D/DOX) had already decomposed into small debris, while spherical structures with intact morphology were observed in pH 7.4 buffer. It was inferred that the pCBMA(CD-D/DOX) showed effective stimuli-responsive performance, which was attributed to the reversible charge of pCBMA and elimination of electrostatic interaction and π-π stacking between DOX and CD-D. Meanwhile, cleavage of disulfide linkage between CDs and peptide dendrimer under redox condition could also be a key factor for accelerating DOX release from CD-D/DOX. Moreover, drug release behavior of CD-D/DOX at same conditions was also examined (Figure S14). At pH 7.4, more drug (28.7%) was released from the CD-D/DOX than pCBMA(CD-D/DOX) (8.5%) after 24 hours; 24 ACS Paragon Plus Environment

Page 25 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


while DOX release of CD-D/DOX reached 71.1% after 24 h exposure at pH 5.5+10 mM DTT. These results demonstrated that zwitterions could largely stabilize the loaded DOX·HCl in the nanoparticles at physiological condition, but not really influence the drug release at acidic tumor sites. Thus, one may deduce that the pCBMA(CD-D/DOX) could selective release the loaded DOX into pathological organ/tissue where the in vivo high concentration glutathione (GSH) and acidic microenvironment would act as triggering stimuli to ensure tumor-specific drug release and to sharply reduce the side effects of DOX on normal organ/tissue. Due to the encapsulation of CD-D/DOX by the linear polymer pCBMA and the surface modification on CDs, it was necessary to explore whether the pCBMA(CD-D/DOX) maintain the fluorescence properties of the CDs. Firstly, it was found that the pCBMA(CD-D/DOX) could be excited by different wavelength light stimulation (Figure S15), demonstrating that the pCBMA(CD-D/DOX)

maintained

the photoluminescence property of CD as shown in Figure S4. Besides, as shown in Figure

3e,

the

maximum

pCBMA(CD-D/DOX)

(360

excitation nm

and

and 450

emission nm

wavelengths

respectively)

of

the

remained

the

excitation-dependent fluorescent property of the parent CDs. This fluorescent property endowed the bioimaging function of the pCBMA(CD-D/DOX). Protein adsorption of the pCBMA(CD-D/DOX). Protein adsorption is a serious problem hindering long-circulation of the nanocarriers in blood streams, by accelerating the clearance of nanoparticles by mononuclear phagocyte system and 25 ACS Paragon Plus Environment


Page 26 of 50

leading to serious inflammatory response.31 Zwitterions have been well known as anti-fouling coatings to prevent formation of biomolecular coronas which were proposed to be universally produced within the broad biological environments.32 To examine the anti-protein adsorption capability of the pCBMA(CD-D/DOX), bovine serum albumin (BSA, 66 kDa) was selected as a model protein to mimic the protein-rich environment in blood. As shown in Figure 3f, the pCBMA(CD-D/DOX) exhibited low protein adsorption amounts during the 4 h experimental time (8.1 %), as comparing with our previously reported micelle systems20 and other works33. This remarkably suppressed protein adsorption given by pCBMA endowed the pCBMA(CD-D/DOX) excellent anti-fouling property, thus prolonged cycle time in blood and increased possibility of the pCBMA(CD-D/DOX) enrichment at tumor sites could be ensured. Cytotoxicity

of

CDs,

peptide

dendrimer,

DOX·HCl,

pCBMA

and

pCBMA(CD-D/DOX). Low toxicity and great biocompatibility are considered as the primary requirements of the nanocarriers for biomedical applications in living bodies. As the main building blocks of pCBMA(CD-D/DOX), CDs, peptide dendrimer (D-S-S-D) and pCBMA were evaluated by CCK-8 assay to investigate their in vitro cytocompatibility to the 4T1 and HepG2 cells. The pCBMA showed good biocompatibility among the test material concentrations (0.01-5 μg/mL) as more than 80% viable cells were detected after co-culture with 4T1 or HepG2 cells (Figure 4). The 4T1 cell survival rates of the CDs, D-S-S-D groups were about 100% after exposure for 48 h in various concentrations (0.01-5 μg/mL), indicating the negligible 26 ACS Paragon Plus Environment

Page 27 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


cytotoxic effect of these basic components (Figure 4a). Notably, as for HepG2 cells, more than 120% cell survival rates were observed in the CDs groups, which confirmed good biocompatibility of CDs (Figure 4b). After excluding the cytotoxicity of CDs, peptide dendrimers and the pCBMA, we evaluated the tumor cell growth inhibition effect of the pCBMA(CD-D/DOX) in comparison to DOX·HCl. In general, the two cell lines displayed a concentration-dependent cell viability manner. The viability of 4T1 cells incubated with pCBMA(CD-D/DOX) at low DOX dose (0.01, 0.1 μg/mL) were almost 100%, while it plunged to 42% and 1% when the DOX concentration increased to 0.5 μg/mL and 1 μg/mL (Figure 4a). The free DOX·HCl exhibited slightly higher toxicity at the same DOX concentration than pCBMA(CD-D/DOX), which was due to the reason that free DOX·HCl was easier to penetrate into tumor cells and nuclei to induce apoptosis than the loaded ones.34 What’s more, similar results could be found in Figure 4b, where the cytotoxicity of DOX·HCl and pCBMA(CD-D/DOX) against HepG2 cells was examined. in vitro fluorescence imaging and endosomal escape. Cellular uptake behavior of the pCBMA(CD-D/DOX) was first measured by flow cytometry. As shown in Figure S16, more pCBMA(CD-D/DOX) were uptaken by 4T1 cells when the incubation time increased from 2 h to 16 h, which was proven by the significantly increased intracellular fluorescence signal. It was obvious, there were 89% cells with internalized pCBMA(CD-D/DOX) after co-incubation for 2 h, and almost all the cells (99%) internalized the treated DOX-containing nanoparticles after 16 h.



Page 28 of 50

Since the CDs possessed fluorescent properties, the in vitro fluorescence imaging capability of pCBMA(CD-D/DOX) was studied by CLSM. As shown in Figure S17, the red and blue fluorescence in the 4T1 cells which corresponded to the intracellular CDs and DOX was excited by 405 nm and 488 nm wavelength lasers respectively. At 2 h, clear fluorescence signals both on CDs and DOX channels in cells were observed, indicating the pCBMA(CD-D/DOX) were cellular internalized and distributed in cytoplasm. Increasing the incubation time to 6 h, more pCBMA(CD-D/DOX) accumulated into cells. It was worth to mention that both CDs and DOX signals were detected not only in cytoplasm, but also in nuclei, which further indicated that the loaded DOX in pCBMA(CD-D/DOX) was released and transported into nuclei. This intercellular DOX trafficking tendency became much more obvious at 16 h. Therefore, one could conclude that the novel versatile pCBMA(CD-D/DOX), containing fluorescent CDs and anti-cancer drug DOX·HCl, holds great potential for applications in biomedical domains as effective theranostic platform. Endosomal escape plays an important role to assist drug transportation to the nucleus after endocytosis. In order to further confirm the effective endosomal escape of the pCBMA(CD-D/DOX), we stained the acidic organelles in 4T1 cells with Lysotracker® green, which was presented as green fluorescent signal. As shown in Figure 5, from 0.5 h to 2 h, more CDs (blue) and DOX (red) were observed in cells and concentrated in the acidic organelles. By prolonging incubation time to 4 h, the DOX signal that appeared outside the lysosome and located at the nuclei strongly suggested that the pCBMA(CD-D/DOX) realized endosomal escape and 28 ACS Paragon Plus Environment

Page 29 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


efficient drug transportation into the nuclei. The localization of CDs, DOX and lysosome could be clearly seen in the pixel intensity profiles (Figure 5, right panel). It showed the overlap of DOX and lysosome increased from 0.5 h to 2 h, but dramatically decreased afterwards, which indicated effective endosomal escape of the drug. Theoretically, this phenomenon was attributed to the protonation of the guanidine groups in the peptide dendrimer functionalized CDs at acidic compartments (endo-lysosomes). The proton sponge effects of the peptide dendrimer here guaranteed the effective endosomal escape and fast drug release at low pH and high GSH concentration environment. in vivo biodistribution. Encouraged by the excellent in vitro antitumor activities of the pCBMA(CD-D/DOX), we further studied their therapeutic efficiency in vivo. Prior to assessing the tumor growth inhibition efficacy, biodistribution of the pCBMA(CD-D/DOX) in vivo was evaluated. In general, pCBMA(CD-D/DOX) was intravenously injected into BALB/c mice bearing subcutaneous mammary carcinoma (loaded DOX concentration was normalized to be 3 mg/kg) and their distribution in main organs (heart, liver, spleen, lungs, kidney) and tumors over 48 h were monitored. As shown in Figure 6a and c, it was obvious that the DOX fluorescent signals were mainly concentrated at tumor, liver and kidney. From 6 h to 24 h and 48 h, the DOX fluorescent signals at tumors displayed a time-dependent increase behavior. More DOX accumulated in tumors as the circulation time increasing, indicating that the pCBMA(CD-D/DOX) indeed targeted the tumor sites. In addition, fluorescent signal intensity at both the liver and kidney showed a remarkable decrease at 48 h, even 29 ACS Paragon Plus Environment


Page 30 of 50

though related signal intensity increased from 6 h to 24 h, indicating that the pCBMA(CD-D/DOX) were gradually eliminated by reticuloendothelial system (RES)-rich organs. To further observe the DOX permeability and retention in tumor sites, cryo-sections of the tumors at different time points were prepared and imaged by CLSM (Figure 6b). The frozen section analysis results were highly consistent with the fluorescent images of ex vivo tissues. In particular, the strongest red fluorescence which represented the largest quantity of DOX accumulation was observed at 48 h. On the other hand, biodistribution of the fluorescent visible CDs was studied (Figure S18), since the CDs have been used to monitor intracellular trafficking of the ternary nanoparticle system (Figure 5) in our work. It has been reported that the CDs in small size

(5~10

nm)

without

any

conjugation

with

other

macromolecular

components/particles could be rapidly cleared within several hours35-36. In this work, the time-dependent accumulation of the injected CDs (~3 nm), in the form of either free CDs or pCBMA(CD-D/DOX), concurred well with these previous reports. Average fluorescent signal (counts) in the CD channel showed a fast reduction within 8 hours. In particular, no fluorescent signal could be detected in the tumors at 8 hours post-administration. Taking an insight into these results, our ternary nanoparticle system enabled the high-level and long-duration accumulation of the therapeutic agents in the tumors as well as fast clearance of the carrier components. All these are exactly what the carrier-assisted chemotherapy required. in vivo pharmacokinetic study. To investigated whether the pCBMA(CD-D/DOX) could prolong drug circulation time in blood, the in vivo pharmacokinetics was 30 ACS Paragon Plus Environment

Page 31 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


performed. The plasma drug concentration-time profiles after administration were shown in Figure 7a and the pharmacokinetic parameters were listed in Figure 7b. The half-life time (t1/2) and AUC (area under the curve) of DOX·HCl were 0.3 h and 7.99 respectively. Encouragingly, the half-life time of pCBMA(CD-D/DOX) increased to 8.69 h (28.9 times) and the AUC raised to 161.80 (20.1 times). This phenomenon could be explained by the anti-protein adsorption ability of the zwitterionic coating (pCBMA) (Figure 3f). The pharmacokinetic results highly demonstrate that the pCBMA(CD-D/DOX) with prolonged circulation time in blood could greatly promote drug accumulation at tumor sites. in vivo therapeutic efficacy. Subsequently, tumor growth inhibition ability of the pCBMA(CD-D/DOX) in vivo was investigated in detail and BALB/c mice bearing subcutaneous mammary carcinoma were selected as model animals. When the tumor volume reached about 100 mm3, the tumor-bearing mice were divided into three groups (n = 5) randomly and were intravenously administrated with saline, DOX·HCl, pCBMA(CD-D/DOX) at day 3, 6, 9 ,12. The DOX concentration of DOX·HCl and pCBMA(CD-D/DOX) was 3 mg per kg of body weight and the volume of saline was 200 μL. Both body weight and tumor size of each mouse were recorded every three days. As the control, tumor volume of the saline group exhibited a fast growth rate (Figure 8a). However, it was noteworthy that the tumor growth rate of pCBMA(CD-D/DOX) group was very slow during the whole experimental period, and the tumor volume remained relatively small (only 246% of initial tumor volume) after four injections (day 12). On the 15th day after treatment, tumor volume of the 31 ACS Paragon Plus Environment


Page 32 of 50

DOX·HCl and pCBMA(CD-D/DOX) groups increased to be 1275% and 516% respectively, while the tumor volume of the saline group rapid soared to 2248%, as compared with the initial tumor volume. The representative photos of excised tumors from the three groups were exhibited in Figure 8b, where the smallest tumor volume of the pCBMA(CD-D/DOX) group was directly visualized, further indicating the better tumor growth inhibition ability of the pCBMA(CD-D/DOX) than DOX·HCl. Compare with saline group, pCBMA(CD-D/DOX) group showed a higher tumor inhibition rate (75.4%) than DOX·HCl group (42.3%) after 15 days’ treatment (Figure S19). Moreover, as compared with the other two groups, the DOX·HCl group showed slower body weight increase which implied serious side effects of free drug DOX·HCl to the tumor-bearing mice (Figure 8c). Histological and immunohistochemical studies. Immunohistochemical and hematoxylin and eosin (H&E) studies were carried out to verify the anticancer activities of each group (Figure 8d). The pCBMA(CD-D/DOX) group showed the largest necrotic area in H&E stained slices of tumor tissue. Moreover, the pCBMA(CD-D/DOX) dramatically reduced the CD31-positive vessels (brown) growth and Ki-67-positive (brown) tumor cell proliferation, compared with the free drug DOX·HCl (Figure 8d). At the same time, the drug induced cell apoptosis was visualized in TUNEL staining results. Unlike the free drug DOX·HCl group, more TUNEL positive signal was observed in pCBMA(CD-D/DOX) group, revealing the better tumor inhibition ability of the pCBMA(CD-D/DOX) than the DOX·HCl. To demonstrate corresponding results clear, the ratios of CD31-positive, Ki-67-positive 32 ACS Paragon Plus Environment

Page 33 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and TUNEL positive areas to the total area of each group were calculated to present underlying mechanism of tumor growth inhibition by the tumor microvessel density (MVD), Ki-67-positive OD (optical density) and TUNEL positive ratio analysis, respectively (Figure S20). Obviously, the pCBMA(CD-D/DOX) group showed less microvessel density and cell proliferation in tumors, on the contrary, less cell apoptosis was found in the DOX·HCl group when the tumor-bearing mice were treated with the same dose of DOX. In addition, the hematoxylin and eosin (H&E) staining of the main organs was used to determine possible pathological changes after drug administration (Figure 8e). Obviously, pulmonary metastasis was found in saline group (marked by an arrow). However, the DOX·HCl and pCBMA(CD-D/DOX) groups only exhibited very few metastasis sites. Meanwhile, the pCBMA(CD-D/DOX) group showed the minimal side effect on the main organs while severe heart damage was observed in DOX·HCl group (pointed by arrows). It was concluded that the pCBMA(CD-D/DOX) could dramatically improve the anti-tumor ability of the therapeutic agents and realize better therapeutic effects as well as reduce the possible toxic side effects in vivo. CONCLUSION In summary, we have developed a specially designed zwitterionic nanoplatform [pCBMA(CD-D/DOX), ~180 nm] for cancer therapy. The peripheral polyzwitterion “coat” (pCBMA) ensured safe delivery of this nanocarrier system in blood circulation owing to its good antifouling property of the zwitterionic structures, which were 33 ACS Paragon Plus Environment


Page 34 of 50

obviously capable to resist nonspecific protein adsorption. More importantly, the zwitterions can also answer to the surrounding pH conditions to expose the positively charged peptide dendrimers, and thereby facilitate cellular uptake. Existence of the acidic compartments and high redox level inside tumor cells offered the facile way to disintegrate the pCBMA(CD-D/DOX), further providing the vast possibility to accomplish tumor site-specific DOX release and high efficient intracellular trafficking toward nuclei. Moreover, the photoluminescence property of the CDs endowed the pCBMA(CD-D/DOX) system with biological imaging potential. Both in vitro and in vivo studies verified the high anti-tumor activity of the pCBMA(CD-D/DOX) (relative tumor volume was 40.5% and 23.0% of the DOX·HCl and Saline groups). Encouragingly, the pCBMA(CD-D/DOX) reduced serious side effect of the free drug on healthy tissue markedly. These results suggest that our strategy possess high potential in developing intelligent drug delivery systems for clinical uses and possess huge value in developing versatile nanoplatforms for desired applications in biomedical areas.


Page 35 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FIGURES

Figure 1. Characterization of CDs. (a) Size distribution. Insert: image of CDs suspension. (b) TEM and (c) HRTEM images. Scale bars indicate 10 nm and 5 nm respectively.

(d)

FTIR

spectrum.

(e)

Excitation

wavelength-dependent

fluorescence spectra.



Page 36 of 50

Figure 2. (a) Zeta potential of CD, CD-SH, CD-D, CD-D/DOX in pH 7.4 buffer solutions. (b) Zeta potential of pCBMA in pH 7.4, 6.8 and 5.5 buffer solutions (means ± SD, n = 3). (c) Size distribution and (d) SEM image, (e) AFM image for 3D architectures of the pCBMA(CD-D/DOX), (f) the size profile along the red line and (g) 3D view of the pCBMA(CD-D/DOX).


Page 37 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. (a) zeta potential changes of the pCBMA(CD-D/DOX) during 4 h incubation in pH 7.4, 6.8 and 5.5 buffer solutions (means ± SD, n = 3). (b) DOX release profiles of the pCBMA(CD-D/DOX) in pH 7.4, and 5.5 with 10 mM DTT at 37 °C (means ± SD, n = 3, **p

Detachable Polyzwitterion-Coated Ternary Nanoparticles Based on

Recommend Documents