Development of Zwitterionic Polypeptide Nanoformulation with High

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Development of Zwitterionic Polypeptide Nano-formulation through Charge Attraction Assembly for Targeted Drug Delivery Weifeng Lin, Guanglong Ma, Zhe-Fan Yuan, Haofeng Qian, Liangbo Xu, Elie Sidransky, and Shengfu Chen Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00851 • Publication Date (Web): 22 Jun 2018 Downloaded from http://pubs.acs.org on June 24, 2018

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Development of Zwitterionic Polypeptide Nano-formulation with High Doxorubicin Loading Content for Targeted Drug Delivery Weifeng Lina, †, Guanglong Maa, †, Zhefan Yuana, Haofeng Qiana, Liangbo Xua, Elie Sidranskyc, and Shengfu Chen a,b* a Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China. b Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, Jiangsu Key Laboratory of Biomedical Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210046, China. c Department of Materials Science and Engineering, A. James Clark School of Engineering, University of Maryland, College Park, Maryland 20740, United States. * To whom correspondence should be addressed: E-mail: [email protected]; †

These authors contributed equally to this work.

Abstract Lots of attention has been drawn to targeted nano-drug delivery systems due to their high therapeutic efficacy in cancer treatment. In this work, doxorubicin (DOX) was incorporated into a zwitterionic arginyl-glycyl-aspartic acid (RGD)-conjugated polypeptide by an emulsion solvent evaporation technique with high drug loading content (45%) and high drug loading efficiency (95%). This zwitterionic nano-formulation showed excellent colloidal stability at high dilution and in serum. The

pH-induced

disintegration

and

enzyme-induced

degradation

of

the

nano-formulation were confirmed by dynamic light scattering (DLS) and gel permeation chromatography (GPC). Efficient internalization of DOX in the cells and high antitumor activity in vitro was observed. Compared with free drug, this nano-formulation showed higher accumulation in tumor and lower systemic toxicity in vivo. The DOX-loaded zwitterionic RGD-conjugated polypeptide vesicles show potential application for targeted drug delivery in the clinic. Keywords: Zwitterionic; Polypeptide; Doxorubicin; Drug delivery. 1

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Introduction Cancer is one of the most prevalent diseases in modern society and become a heavy burden on many countries’ healthcare systems.1 Chemotherapy is still the most popular treatment for many types of cancers. However, the systemic toxicity and adverse effects of anticancer drugs restrict their extensive applications.2-7 Therapeutic agents in conjunction with nano-carriers, such as liposomes,8-10 vesicles,11-13 micelles,14-17 and inorganic materials18-19 result in better pharmacokinetic properties, enhanced tumor accumulation through the enhanced permeability and retention (EPR) effect20 or targeting group (such as folic acid and arginine-glycine-aspartic acid (RGD))21-23, and fewer side-effects on healthy tissues and organs. However, conventional carriers possess the drawbacks of low drug-loading capacity (generally not higher than 10%), low drug-loading efficiency, and low metabolizability through natural pathways. This may cause long-term systemic toxicity after repeated administrations and drive up the cost of therapy, thereby limiting their clinical applications.24 Recently, several nanodrug formulations have been developed to solve these problems.25-32 For example, Cheng and co-workers reported about nanoparticles that could load the dimerized drug with over 50% drug loading content and nearly 100% drug loading efficiency.33 This nano-formulation showed excellent stability under physiological conditions without triggering, while thiol triggering resulted in a controlled release of its authentic form of drug. Lecommandoux and coworkers found that

the

vesicles

of

poly(trimethylene

carbonate)-b-poly(L-glutamic

acid)

(PTMC-b-PGA) can load DOX with a drug loading content of 47% and a loading efficiency between 50 and 70% w/w. Besides, it showed accelerated drug release in acidic conditions while stable under storage conditions.13 Zwitterionic polymers have been found to possess excellent resistance to nonspecific interaction with proteins, cells, and bacteria.34-40 Biodegradable zwitterionic polypeptides, composed of either alternating or randomly mix charged certain natural amino acids, are unique biomimetic materials,41-44 which resemble the amino acid composition on the surface of cytoplasm proteins with resistance to 2


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nonspecific interaction.45 As glutamic acid (E) and lysine (K) pair is the most abundant pair in cytoplasm proteins, albumin, which is also an EK abundant protein, has

longer

half-life

circulation

time46

than

any

synthetic

nanoparticles.

Albumin-bound paclitaxel (Abraxane®) is one of the most successful nano-drug formulations for cancer treatment. Thus, it is expected to mimic albumin by EK polypeptides to load hydrophobic anti-cancer drug to achieve high antitumor efficacy and low systemic toxicity. Recently, our group reported that a polypeptide composed of a hydrophobic block and a zwitterionic block formed micelles in aqueous solution.39 DOX could be loaded into the micelles through hydrophobic interaction. Because of the zwitterionic polypeptide shell, the micelles showed both low nonspecific protein adsorption and cell uptake, with relative low drug loading content (8.7%). In this work, RGD-conjugated polypeptide composed of E and K was synthesized for encapsulation positively charged DOX in negatively charged EK peptide by electrostatic attraction through tuning the ratio of E to K over 1:1. A DOX-loaded vesicle with high drug loading (45%) and high drug efficiency (95%) was prepared by an emulsion solvent evaporation technique (Scheme 1). The stability in different conditions, in vitro release behaviors, enzymatic degradation, cell uptake, and anticancer efficacy of this nano-formulation were investigated. Furthermore, the biodistribution and antitumor efficacy of this nano-formulation in mice were also investigated.

Materials and Methods Materials Nε-Carbobenzyloxy-L-Lysine (H-Lys(Z)-OH, ≥98%) and γ-Benzyl-L-Glutamate (Glu(OBzl), ≥98%) were purchased from GL Biochem Co., Ltd. Triphosgene (99%) was purchased from Adamas Reagent Co., Ltd. Maleic anhydride, n-hexylamine and triethylamine (TEA, 99.5%) were purchased from Aladdin Reagent Co., Ltd. Extra dry dimethylformamide (DMF) was purchased from Alfa Aesar. Tetrahydrofuran (THF), n-hexane, and chloroform were purchased from Sinopharm Chemical Reagent 3


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Co., Ltd. THF was dried by refluxing over Na metal under an argon atmosphere and distilled immediately before use. Cyclo(Arg–Gly–Asp–D-Phe–Cys) (c(RGDfC), 95%) was purchased from Chutai Biotechnology Co., Ltd. 33 wt.% HBr/HOAc solution was purchased from Sigma-Aldrich Co., Ltd. Doxorubicin hydrochloride (DOX·HCl, 99%) was purchased from Taizhou XinFangXiang Chemical Co., Ltd. Doxorubicin hydrochloride was converted to its free base form (DOX) by reacting with 3 molar equivalents of triethylamine. N-carboxyanhydride (NCA) of Glu(OBzl) and NCA of Lys(Z)

were synthesized according to procedures described previously.47

As shown in Scheme S1, the poly (L-glutamic acid-co-L-lysine) (PMIX) with different glutamic acid to lysine ratios was synthesized according to our previous study.47 Gel permeation chromatography (GPC) for PMIX was performed in aqueous sodium nitrate (NaNO3) solution (0.5 mol/L) at 40 oC using Waters GPC system equipped with Waters ultrahydrogel columns and a Waters refractive index detector. The molecular weights and the molecular weight distribution of the polymer samples were calculated on the basis of poly(ethylene glycol) calibration. Synthesis of c(RGDfC) Modified PMIX In the procedures shown in Scheme 2, PMIX (50 mg) was dissolved in 1 mL 50 % DMSO aqueous solution, and 0.1 M NaOH was added until the solution reached a pH of 8. Maleic anhydride (with a 2% molar ratio of the amino group in PMIX) was dissolved in 20 µL of DMSO, after which the prepared DMSO solution was added dropwise to the PMIX solution under a nitrogen (N2) atmosphere. After mixing, the solution was stirred for 6 hours at ambient temperature. This solution was precipitated with excess methanol after concentration. Then the precipitate (PMIX-MA) was washed with excess ether and finally dried under vacuum overnight. Then, PMIX-MA was dissolved in double distilled water under a nitrogen atmosphere. The c(RGDfC) aqueous solution was added dropwise to the PMIX-MA solution under N2 protection; The michael addition reaction was supposed to complete overnight, and to remove the residual reactants, the solution was then placed into a 10 kDa molecular-weight-cutoff Amicon Ultra centrifugal filter device (Sigma-Aldrich) for centrifugation. The washing procedure was repeated until no c(RGDfC) was 4


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detected by DTNB (5,5’-Dithiobis(2-nitrobenzoic acid)) method. c(RGDfC) modified PMIX was obtained after lyophilization and kept in -20 oC. Drug Encapsulation PMIX was dissolved in carbonate buffer (pH 10.0, 50 mM). Varying amounts of DOX were dissolved in chloroform, and these samples were then emulsified with PMIX solution using a sonicator for 30 s to form a stable emulsion. Subsequently, chloroform was evaporated with stirring at 27.5 °C for 4 h. Next, the solution was centrifuged using Millipore (cutoff = 10000 Da, 6000 rpm), then the solution was diluted with PBS (pH 7.4) and centrifuged several times until the pH of the solution was 7.4 and stored at 4 °C. Drug loading content (DLC) and drug loading efficiency (DLE) of DOX encapsulated in PMIX were determined by fluorescent spectrometry. A calibration curve was obtained using different concentrations of DOX·HCl in DMSO/H2O (4/1, v/v). DLC and DLE were calculated according to the following formulas, respectively. DLC (wt %) = (weight of encapsulated drug/total weight of encapsulated drug and polymer) × 100 DLE (%) = (weight of loaded drug/weight of drug in feed) × 100 Particle Size and Zeta Potential The average diameters and zeta potential of the PMIX-DOX were measured by Zetasizer Nano-ZS (Malvern Instruments Ltd., Malvern, UK) with a red laser light (wavelength λ = 632.8 nm) and the scattering angle at 173°. Transmission electron microscopy (TEM) samples were prepared by floating the carbon-coated copper grid on a drop of a solution of PMIX-DOX, and then been dried. TEM analysis was performed by JEM-1200EX TEM (JEOL Ltd., Japan) operating at an accelerating voltage of 80 kV. Evaluation of the Stability of PMIX-DOX The stability of PMIX-DOX was evaluated by measuring their sizes under different conditions. The size of PMIX-DOX particles after diluting with PBS, different pH buffers, 100% FBS was tested at 37 °C using dynamic light scattering (DLS) 5


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measurements. The FBS solution was filtrated through100 nm filter before use. In Vitro Drug Release To determine the release profiles of DOX, 0.5 mL of PMIX1.2/1-DOX solution was transferred into a dialysis bag (MWCO 3500 Da). The release experiment was carried out by placing the dialysis bag into 10 mL of three release media (pH 7.4 PBS, pH 5.0 sodium acetate buffer, pH 5.0 sodium acetate buffer with 50 U/mL trypsin) at 37 oC with constant shaking at 200 rpm. At predetermined time intervals, 0.8 mL of the medium outside was taken and tested using fluorescence spectrometer. Accordingly, the releasing media was replenished with 0.8 mL of fresh medium. Then the concentration of released DOX was monitored. Enzymatic Degradation of PMIX Polymer First, PMIX was dissolved in pH 5.0 sodium acetate buffer or pH 5.0 sodium acetate buffer with 50 U/mL trypsin at a concentration of 3 mg/mL. The solutions were placed in a shaker at 37 °C for 12 hours and then passed through a 0.22 µm filter for GPC detection. The mobile phase was 0.5 M NaNO3, the flow rate was 0.5 mL/min, and the column temperature was set at 40 °C with a differential refractive index detector. Cell Viability Assay The cell viability of PMIX, PMIX-DOX, and RGD-PMIX-DOX were evaluated by MTT assays. U87 cells (Uppsala 87 Malignant Glioma) were seeded into 96-well plates at a density of 10000 per well in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS under 5% CO2 for 24 h. Next, cells were exposed to DMEM medium containing different concentrations of PMIX or different concentrations of DOX for 24 h. Then the medium was replaced by 180 µL growth medium containing 20 µL MTT solution (5 mg/mL in PBS). After incubation for another 4 h, the MTT-containing medium was replaced by 200 µL DMSO to dissolve the obtained crystals. The absorbance was measured at the wavelength of 570 nm. The relative cell viability (%) was determined by comparing the absorbance with control wells at 570 nm. Cellular Uptake 6


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The cellular uptake of PMIX-DOX or RGD-PMIX-DOX was performed by inverted fluorescence microscope and flow cytometry. For inverted fluorescence microscope, U87 cells were seeded in 24-well plates in DMEM with 10% FBS, under 5% CO2 at 37 °C for 24 h. Culture medium was discarded, and 1.0 mL of free DOX, PMIX-DOX, or RGD-PMIX-DOX (DOX concentration, 5 µg/mL) solution in DMEM were added to each well. After being incubated at pre-set time points, the medium was removed. Then the cells were rinsed three times with PBS and stained with Hoechst33342. The cellular subcellular distribution was photographed by inverted fluorescence microscope. For flow cytometry, U87 cells were seeded in 6-well plates in DMEM with 10% FBS, under 5% CO2 at 37 °C for 24 h. Culture medium was removed, and 1.0 mL of free DOX, PMIX-DOX, or RGD-PMIX-DOX (DOX concentration, 5 µg/mL) solution in DMEM were added into each well. After being incubated at pre-set time points, the medium was removed. Then, the cells were rinsed three times with PBS, harvested by trypsin treatment and resuspended in 0.4 mL of PBS, and analyzed using a flow cytometer (BD FACSEALIBUR, San Jose, USA). Biodistribution of PMIX-DOX All animal experiments were conducted in accordance with guidelines established by the Institute for Experimental Animals of Zhejiang University. Healthy female nude mice were purchased from the animal center of Zhejiang Academy of Medical Sciences. Subcutaneous tumor models were established in BALB/c Nude Mouse by subcutaneous injection of 1 × 107 U87 cells in the flank region. The tumor-bearing nude mice were randomly divided into two groups with five mice in each group when the tumor size reached 100 mm3. Free DOX and RGD-PMIX-DOX were injected into U87 tumor-bearing BALB/c nude mice via the tail vein at a dosage of 10 mg of doxorubicin per kg of body weight. At predetermined time intervals, the mice were sacrificed and the tumors and the normal organs were excised for ex vivo imaging. In Vivo Antitumor Therapy The establishment of animal tumor model is the same as biodistribution experiment. The tumor-bearing nude mice were randomly divided into three groups with five 7


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animals in each group, when the tumor size reached 100 mm3. Free DOX, RGD-PMIX-DOX and PBS were injected into U87 tumor-bearing BALB/c nude mice via the tail vein at a dosage of 5 mg of doxorubicin per kg of body weight at an interval of 2 days (day 0, 2, 4, and 6). The length and width of the tumors and the body weight of mice were recorded at regular intervals. Tumor volume (V) was calculated using the following formula: V = length × width2/2. Statistical Analysis Student’s t-test was performed to determine the statistical significance, and p-values less than 0.05 were considered to define statistically significant. All results were expressed as mean ± standard deviation (S.D.).

Results and Discussion Synthesis and Characterization of PMIX The

peptide

poly[Glu(oBzl)-co-Lys(Z)]

was

synthesized

via

ring-opening

polymerization (ROP) of Glu-NCA and Lys-NCA initiated by n-hexylamine. After removing the protective groups poly[Glu(oBzl)-co-Lys(Z)] using 33 wt.% HBr/HOAc solution, the zwitterionic polypeptide PMIX was obtained (Scheme S1). The characteristic peaks at i, i” (4.95 ppm, –CH2C6H5–) and j, j” (7.21 ppm, –C6H5) indicated the polymerization reaction successfully proceeded (Figure 1a). The disappearance of the phenyl peaks at j, j” (7.21 ppm, –C6H5) after fully deprotection indicated that poly[Glu(oBzl)-co-Lys(Z)] was completely converted into zwitterionic polypeptide poly (L-glutamic acid-co-L-lysine) (PMIX) (Figure 1b) with the deprotection procedure. Additionally, the molecular weight and molecular weight distribution (polydispersity index, PDI) of PMIX were measured by GPC and the molar ratios of Glu and Lys were calculated by comparing the ratio of intensity of peak e and peak h (Table 1). The results showed that the number molecular weights of PMIX were 7 ~13 kDa and the PDIs varied from 1.3 to 1.7. Preparation of PMIX-DOX Nanoparticles The carboxyl groups on the side chain of glutamic acid were ionized by dissolving the PMIX in carbonate buffer (pH 10). Then solution of doxorubicin in chloroform was 8


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added to the PMIX aqueous solution. The composition of PMIX-DOX can be expressed with the feed [DOX]:[extra COO-] molar ratio, which was chosen to be 1:5, 1:2 and 1:1. The stability of PMIX-DOX nanoparticles in different feed ratio after 4 h evaporation and ultrafiltration centrifugation were shown in Table 2. It can be seen that the PMIX-2/1-DOX and PMIX-1.5/1-DOX are no longer stable with increased drug feeding ratio, while the drug-loaded vesicle of PMIX-1.2/1-DOX was stable at all feeding ratios. It is clearly show that there is limitation of mass ratio between hydrophobic DOX and hydrophilic EK peptide since hydrophilic EK peptide need to cover the hydrophobic sub-group in DOX. More extra negative charge only slightly increased the encapsulation of positive charge DOX through charge attraction. Furthermore,

the

drug

encapsulation

efficiency

and

loading

content

of

PMIX-1.2/1-DOX were very high and reached to 95% and 45 wt%, respectively (where the feeding ratio of [DOX]/[extra COO-] was 1:1). Only 5% extra COO- was left in PMIX-1.2/1-DOX vesicles according to the 1:1 feed ratio of [DOX]:[extra COO-] and 95% drug loading efficiency. This means that the molar ratio of negatively charged carboxyl groups to positively charged amine in PMIX-1.2/1-DOX vesicles is about 1.2:1.19 at pH 7.4, indicating that the PMIX-1.2/1-DOX is very close to zwitterionic condition and will show good resistance to nonspecific protein adsorption. Thus, the PMIX-1.2/1-DOX was the key candidate in the following investigation, and the following PMIX-1.2/1-DOX in this paper refers to PMIX-1.2/1-DOX with a feeding ratio1:1 of [DOX]/[extra COO-]. The hydrodynamic diameter of PMIX-1.2/1-DOX was measured using DLS (Figure 2a). The diameter of PMIX-1.2/1-DOX was 163.5 ± 18.5 nm, which could leak out preferentially into the tumor tissue through permeable tumor blood vessels. The TEM micrograph showed that PMIX-1.2/1-DOX have collapsed vesicles morphology due to drying-artifacts during the preparation of TEM samples (as shown in Figure S3). The zeta potential of PMIX-1.2/1-DOX was close to charge neutral, about -10 mV from pH 5 to pH 7.4 (Figure 2b), which agrees well with the characteristic of most zwitterionic polymer coated surfaces/or particles. Only when the pH value was below 4.5, the zeta potential of PMIX-1.2/1-DOX changed to 9


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positive range, which indicates the protonation of -COO- is the key reason for the charge reversal since the charge transition pH value is close to the range of pKa of carboxyl group. Stability of PMIX-DOX at High Dilution, in Serum and in Acidic Condition Polymeric nanoparticles after intravenous injection would face two challenges. One is the issue of dissociation at high dilution after injection into the bloodstream, which may induce a premature release of drug from the vehicle.48-50 The other is the serum protein adsorption, which causes the risk of aggregation of nanoparticles during blood circulation.51-53 The stability of PMIX1.2/1-DOX under high dilution was recorded by DLS measurements (Figure 3). In particular, the size of PMIX1.2/1-DOX showed no increase after 400-fold dilution (0.0025 mg/mL), a slight increase after 1000-dilution (0.001 mg/mL) while maintaining a low PDI, but a huge increase from 160 nm to 600 nm after 5000-dilution (0.0002 mg/mL). These results indicate the drug-loaded PMIX-DOX is stable against dilution. 100% FBS was chosen to evaluate the stability of PMIX-DOX in serum. As can be seen from Figure 4, the size of vesicle only varied within 6 nm in 100% FBS over 24 h and much less than the dramatic size increase of protein adsorbable gold nanoparticles54, which clearly suggests that the nonspecific adsorption of serum protein on the drug-loaded PMIX-DOX vesicle does not occur due to the zwitterionic nature of the PMIX1.2/1-DOX vesicle. This high stability of the drug-loaded PMIX-DOX vesicle mainly comes from the charge attraction between positively charged DOX and negatively charged PMIX1.2/1 combining with the hydrophobic interaction of the anthracycline group from DOX. Due to the acidic environment of tumor tissue and endosomes presenting in the biological system, the stability of PMIX1.2/1-DOX at different pH environments was also checked. It can be found that the size of vesicles increased slightly with a decreasing of pH from 7.4 to 6.0; when the pH dropped to 5.0, macroscopic aggregation appeared (Figure 5). By checking the zeta potential, the charge-reversal occurred when the pH was reduced to 4.0 (Figure 2b). This may be due to the fact that as the external pH approaches the isoelectric point of the polymer, the protonation of a few of carboxyl groups causes stronger hydrogen bonds between the vesicles, giving 10


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rise to aggregation, which is in accordance with the mechanism of surface protonation of the zwitterionic nano-drug vehicle (NDV) triggered by pH decrease.55 At the same time, protonation of carboxyl groups also leads to a weakening of the interaction between the polymer and doxorubicin, resulting in a decrease of vesicle stability. In Vitro Release of PMIX1.2/1-DOX To examine the effect of pH and enzyme on in vitro release behavior of DOX, PMIX1.2/1-DOX was placed in different solutions as described before. As shown in Figure 6, DOX releasing from PMIX1.2/1-DOX was relatively slow in pH 7.4 buffer--less than 20% encapsulated DOX release after 24 hours. However, more than 60% DOX was released at pH 5.0 within 24 hours, suggesting that PMIX1.2/1-DOX shows pH-sensitive release behavior. Trypsin, a common digestive enzyme secreted by the pancreas, has also been shown to be expressed in a wide range of cancers, involving in proliferation, invasion, and metastasis of tumor.56-59 After adding trypsin into pH 5.0 buffer, the release of DOX at the first 4 hours almost doubled comparing with pH 7.4 buffer with or without trypsin, which was lower than our expected accelerating rate value. Although the optimal pH of trypsin is 7.8 - 8.5, the precipitation formed by degraded PMIX1.2/1-DOX and trypsin at pH 7.4 progressively decreased the degradation rate of EK peptide in PMIX1.2/1-DOX (Figure S2) since the quick enzymatic hydrolysis of PMIX polypeptides (as shown in Figure 7) resulted in the destruction of the original vesicle structure and reconstitution to form the aggregation. On the other hand, the activity of trypsin is yet inhibited under acidic conditions. Thus, the additional trypsin just reasonably increased the DOX release rate in pH 5.0 buffer. PMIX1.2/1-DOX and RGD-PMIX1.2/1-DOX Internalization The internalization of PMIX1.2/1-DOX and RGD-PMIX1.2/1-DOX was investigated by incubating the drug-loaded vesicles with U87 cells. As shown in Figure 8, the red fluorescence observed from DOX by inverted fluorescence microscope was applied to visualize the cellular internalization of the vesicles. The internalization of PMIX1.2/1-DOX drug-loaded vesicles increased significantly under acidic condition (pH 6.0). Meanwhile, an opposite behavior was seen for free DOX at pH 7.4 It clearly 11


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indicates that the protonation of carboxylic groups on EK peptide can enhance the internalization of PMIX1.2/1-DOX as observed in our other work.55 On the contrary, doxorubicin is an anthracycline consisting of an ionizable primary amine with a pKa value of 8.3. The ratio of non-ionized form to ionized form of the drug would decrease with lowering pH (pH 6.0), resulting in less non-ionized DOX molecules participated in the hydrophobic zone of cell membrane for internalization. Therefore, this finding suggests that the acidic environment of tumor tissue could enhance the internalization of PMIX1.2/1-DOX. In glioblastoma, αvβ3 integrin is over-expressed at the invasive margins of the tumor. RGD has been developed as targeting moiety, which could show preferential binding to αvβ3 integrin, which could effectively achieve the targeting of tumor cells and promote the internalization of drug-loaded vesicles. In order to conjugate targeting group RGD on the polypeptides, PMIX was first reacted with maleic anhydride, which was verified by the appearance of new peaks of double bond protons (at 6.2 ppm and 5.8 ppm, as shown in Figure S1a). According to the ratio of peak areas, 1.5% of the amino groups were modified by maleic anhydride. Then, c(RGDfC) was conjugated onto the PMIX through Michael addition between the double bonds of the maleic acid and the thiol groups of c(RGDfC). The complete disappearance of double bond proton and a new signal of benzene ring peak after the reaction proved a successful modification (Figure S1b). As shown in Figure 8 and 9, higher cell fluorescence intensity with incubation of RGD-PMIX-DOX was observed than those with incubation of PMIX-DOX, which reveals that the RGD modification enhanced cellular uptake of encapsulated DOX in tumor cells lines. From these obtained results, we can see that the the zwitterionic peptides nano-formulation can be stable in complex protein environment, resulting in prolonged circulation time of nano-drug carriers, which is considered to be passively targeting tumor tissue. Additionally, they can also actively target tumor cells due to the tumor-targeting of RGD peptides to the αvβ3 integrin and also the pH sensitive enhancement of internalization. In short, the RGD-conjugated zwitterionic polymers target tumor tissues via both active and passive targeting methods. 12


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In vitro Cell Cytotoxicity Evaluation The relative cytotoxicity of PMIX-1.2 was accessed by MTT cell viability assay on U87 and HEPG2 cells. Figure 10a shows that PMIX reveals low cytotoxicity in both U87 and HepG2 cells, even at concentrations of 0.5 mg/mL, which is well above those used in vitro. The relative cell viability was around 90% after 24 h incubation, implying that zwitterionic polypeptide PMIX-1.2 possessed non-cytotoxicity and excellent biocompatibility under physiological condition. Figure 10b shows that RGD-PMIX1.2/1-DOX exhibits the highest inhibition to the proliferation of U87 and DOX exhibits the lowest inhibition at pH 6.0. These results are in good agreement with the results of cellular uptake of PMIX-DOX in vitro and suggest the RGD-PMIX1.2/1-DOX is much safer and will exhibit high uptake if the tumor tissue becomes acidic. In Vivo Evaluations of Biodistribution and Antitumor Efficacy For biodistribution studies, ex vivo DOX fluorescent imaging of isolated major internal organs (heart, liver, spleen, lungs, and kidneys) and tumors in different time points post-injection, was carried out on nude mice bearing U87 tumors. As can be seen in Figure S4, free DOX was observed mainly in kidneys, suggesting that the free DOX molecules were quickly excreted from kidneys. Such phenomena would cause reduced drug efficacy and side effects on the organs. On the other hand, the enhanced accumulation of DOX in tumor tissue was observed for RGD-PMIX1.2/1-DOX, which might be due to the prolonged circulation time in blood, fast internalization by cancer cells, as well as intracellular environment-responsive (pH and enzyme) drug release of the new formulation. To evaluate the antitumor effects of RGD-PMIX1.2/1-DOX in vivo, a xenograft tumor model of Glioblastoma was established on nude mice (Figure 11a). While a rapid tumor growth was observed in the control group (treated with saline), RGD-PMIX1.2/1-DOX inhibited tumor growth by 60%. Although the tumor growth inhibition was evaluated to be 80% while treating with free DOX, the average body weight of this group of mice declined dramatically by 27 % (as shown in Figure 11b), implying that severe harmful adverse effects were caused by free DOX. On the 13


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contrary, treating with RGD-PMIX1.2/1-DOX, there was substantial increase in body weight as a consequence of natural growth, which is in agreement with the control group Conclusion In

conclusion,

zwitterionic

biodegradable

c(RGDfC)-modified

DOX-loaded

polypeptide vesicles with high drug loading content (45%) and loading efficiency (95%) were prepared using an emulsion solvent evaporation technique. These vesicles showed excellent stability, accelerated intracellular drug release (pH and enzyme-responsive), higher tumor accumulation, and lower systemic toxicity. All these features suggest that the nanodrug formulation would have great potential as a targeted drug delivery for cancer therapy. Acknowledgements

The authors appreciate financial support from the National Nature Science Foundation of China (21674092 and 21474085), the National Development Project on Key Basic Research (973 Project, 2015CB655303), the Ph.D. Programs Foundation of Ministry of Education of China (20110101110034) and Zhejiang Provincial Natural Science Foundation of China (LZ13E030001). Conflicts of interest There are no conflicts of interest to declare.

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Table 1 Composition, molecular weight and polydispersity index (PDI) of PMIX Sample PMIX-2/1 PMIX-1.5/1 PMIX-1.2/1 PMIX-1/1

Feed ratio (Glu/Lys) 2/1 1.5/1 1.2/1 1/1

Actual ratio (Glu/Lys) 2/1 1.56/1 1.25/1 1/1

Mn/(104 g/mol)

PDI

0.9 1.3 0.7 0.8

1.3 1.4 1.3 1.7

Table 2 Stability of different DOX ratio loaded vesicles. Feed ratio [DOX]/[extra COO-] Sample 20% 50% 100% PMIX-2/1 stable unstable unstable PMIX-1.5/1 stable stable unstable PMIX-1.2/1 stable stable stable PMIX-1/1 unstable unstable unstable

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Figures and Captions Scheme 1 Illustrations of doxorubicin encapsulation by zwitterionic polypeptide with targeting moiety. Scheme 2 Synthesis procedure of c(RGDfC) modified PMIX. Figure 1 (a) 1H NMR spectra of poly[Glu(oBzl)-co-Lys(Z)] in DMSO-d6. (b) 1H NMR spectra of PMIX in D2O. Figure 2 Characterization of PMIX1.2/1-DOX nanoparticles. (a) size distribution (b) zeta potential under different pH environments. Figure 3 Size distributions of PMIX1.2/1-DOX nanoparticles before and after dilution by PBS. Figure 4 Hydrodynamic size of PMIX1.2/1-DOX nanoparticles over storage time in 100% FBS at 37 °C (mean ± SD, n = 3). Figure 5 Stability of PMIX1.2/1-DOX nanoparticles under different pH environments. Figure 6 Drug release behavior of PMIX1.2/1-DOX nanoparticles under different conditions. Figure 7 The molecular weight change of PMIX1.2 before and after 12 h incubation in 50 U/mL trypsin (pH 7.4). Figure 8 Inverted fluorescence microscope images showing the U87 Cellular uptake of DOX (A-C), PMIX1.2/1-DOX (D-F), and RGD-PMIX1.2/1-DOX (G-I) under pH 6.0 (a) and 7.4 (b) following 3 h of exposure. Panels A, D, and G show the location of the Hoechst 33342 stained cell nucleus (blue), panels B, E, and H show the location of the DOX (red), and panels C, F, and I show the overlay of DOX and Hoechst 33342 channels. The drug concentration is 5 µg ml−1. Figure 9 (a) U87 cellular uptake after 1 and 6 h of incubation at pH 6.0 and 37 oC, analyzed by flow cytometry with a minimum of 1×104 events. (b) Mean fluorescence intensity located in the U87 cells incubated at pH 6.0 and 37 oC. (black: PBS; blue: DOX,

1

h;

green:

RGD-PMIX1.2/1-DOX,

1

h;

19


DOX,

6

h;

orange:

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RGD-PMIX1.2/1-DOX, 6 h). Figure 10 (a) Relative cell viability of U87 and HepG2 cells after 24 h’s incubation with PMIX-1.2/1. (b) Relative cell viability of U87 and HepG2 cells after 24h’s incubation with RGD-PMIX1.2/1-DOX under pH 6.0 and 7.4 (mean ± SD, n = 5). Figure 11 Relative tumor volume change (a) and body weight change (a) of tumor bearing nude mice after injection of PBS, RGD-PMIX1.2/1-DOX, and Free DOX. ∗ denotes p < 0.05, ∗∗ denotes p < 0.01.

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Scheme 1 Illustrations of doxorubicin encapsulation by zwitterionic polypeptide with targeting moiety.

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Scheme 2 Synthesis procedure of c(RGDfC) modified PMIX.

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(a)

(b) Figure 1 (a) H NMR spectra of poly[Glu(oBzl)-co-Lys(Z)] in DMSO-d6. (b) 1H NMR spectra of PMIX in D2O. 1

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(a)

(b) Figure 2 Characterization of PMIX1.2/1-DOX nanoparticles. (a) size distribution (b) zeta potential under different pH environments.

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Figure 3 Size distributions of PMIX1.2/1-DOX nanoparticles before and after dilution by PBS.

Figure 4 Hydrodynamic size of PMIX1.2/1-DOX nanoparticles over storage time in 100% FBS at 37 °C (mean ± SD, n = 3).

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Figure 5 Stability of PMIX1.2/1-DOX nanoparticles under different pH environments.

Figure 6 Drug release behavior of PMIX1.2/1-DOX nanoparticles under different conditions.

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Figure 7 The molecular weight change of PMIX1.2 before and after 12 h incubation in 50 U/mL trypsin (pH 7.4).

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(a)

(b) Figure 8 Inverted fluorescence microscope images showing the U87 Cellular uptake of DOX (A-C), PMIX1.2/1-DOX (D-F), and RGD-PMIX1.2/1-DOX (G-I) under pH 6.0 (a) and 7.4 (b) following 3 h of exposure. Panels A, D, and G show the location of the Hoechst 33342 stained cell nucleus (blue), panels B, E, and H show the location of the DOX (red), and panels C, F, and I show the overlay of DOX and Hoechst 33342 channels. The drug concentration is 5 µg ml−1. 28


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(a)

(b) Figure 9 (a) U87 cellular uptake after 1 and 6 h of incubation at pH 6.0 and 37 oC, analyzed by flow cytometry with a minimum of 1×104 events. (b) Mean fluorescence intensity located in the U87 cells incubated at pH 6.0 and 37 oC. (black: PBS; blue: DOX,

1

h;

green:

RGD-PMIX1.2/1-DOX,

1

h;

RGD-PMIX1.2/1-DOX, 6 h).

29


DOX,

6

h;

orange:

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(a)

(b) Figure 10 (a) Relative cell viability of U87 and HepG2 cells after 24 h’s incubation with PMIX-1.2/1. (b) Relative cell viability of U87 and HepG2 cells after 24h’s incubation with RGD-PMIX1.2/1-DOX under pH 6.0 and 7.4 (mean ± SD, n = 5).

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(a)

(b) Figure 11 Relative tumor volume change (a) and body weight change (a) of tumor bearing nude mice after injection of PBS, RGD-PMIX1.2/1-DOX, and Free DOX. ∗ denotes p < 0.05, ∗∗ denotes p < 0.01.

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Development of Zwitterionic Polypeptide Nanoformulation with High

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