Erythrocyte Membrane-Wrapped pH Sensitive Polymeric

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Erythrocyte membrane-wrapped pH sensitive polymeric nanoparticles for non-small cell lung cancer therapy Lipeng Gao, Hao Wang, Lijuan Nan, Ting Peng, Lei Sun, Jinge Zhou, Ye Xiao, Jing Wang, Jihong Sun, Weiyue Lu, Lin Zhang, Zhiqiang Yan, Lei Yu, and Yiting Wang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00428 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 6, 2017

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Bioconjugate Chemistry

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Erythrocyte membrane-wrapped pH sensitive polymeric nanoparticles for non-small cell lung cancer therapy Lipeng Gao,†,# Hao Wang,†,# Lijuan Nan,† Ting Peng,† Lei Sun,† Jinge Zhou,† Ye Xiao,† Jing ⊥ Wang,† Jihong Sun,‡ Weiyue Lu,§ Lin Zhang, Zhiqiang Yan,*,† Lei Yu,† and Yiting Wang*,† †

Institute of Biomedical Engineering and Technology, Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, P.R. China. ‡ Department of Radiology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310016, P.R. China. § Department of Pharmaceutics, School of Pharmacy, Fudan University & Key Laboratory of Smart Drug Delivery, Fudan University, Ministry of Education, Shanghai 201203, P.R. China. ⊥ Department of Pharmacy, Shaoxing People’s Hospital, Shaoxing Hospital of ZheJiang University, Shaoxing, Zhejiang 312000, P.R. China. # These authors contributed equally to this work. Corresponding Authors: *E-mail: [email protected] *E-mail: [email protected]

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Abstract The application of nano drug delivery systems (NDDSs) may enhance the effectiveness of chemotherapeutic drugs in vivo. However, the short blood-circulating time and poor drug release profile in vivo are still two problems with them. Herein, by using red blood cell membrane (RBCm) wrapping and pH sensitive technology, we prepared RBCm wrapped pH sensitive poly (L-γ-glutamylcarbocistein)-paclitaxel (PGSC-PTX) nanoparticles (PGSC-PTX@RBCm NPs), to prolong the circulation time in blood and release PTX timely and adequately in acidic tumor environment. The PGSC-PTX NPs and PGSC-PTX@RBCm NPs showed spherical morphology with average sizes about 50 nm and 100 nm, respectively. The cytotoxicity of PGSC-PTX@RBCm NPs was considerably decreased compared with that of PGSC-PTX NPs. PTX release from PGSC-PTX and PGSC-PTX@RBCm NPs at pH 6.5 was remarkably higher than those at pH 7.4, respectively. The PGSC-PTX@RBCm NPs exhibited remarkably decreased uptake by macrophages than PGSC-PTX NPs. The area under curve within 72 h (AUC0-72h) for is significantly higher than PGSC-PTX NPs. The PGSC-PTX@RBCm NPs also showed significantly stronger growth-inhibiting effect on tumor than PGSC-PTX NPs. These results indicated that PGSC-PTX@RBCm NPs have acidic sensitivity of drug release, the characteristic of long circulation, and remarkable effect of inhibiting tumor growth. This study may provide an effective strategy for improving the antitumor effect of NDDS.

Keywords:

Paclitaxel, pH sensitive, red blood cell membrane wrapped, long

circulation, drug delivery Table of Contents graphic (TOC)

Erythrocyte membrane-wrapped pH sensitive polymeric nanoparticles for non-small cell lung cancer therapy



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Introduction Paclitaxel (PTX) is one of the most effective chemotherapeutic drugs and is mainly used to treat lung, ovarian, and breast cancer, etc.1,2 The mechanism of action of PTX is to promote and stabilize microtubules and inhibit the late G2 or M phases of cell cycle, thereby causing cell death.3 Due to the low water solubility of PTX (0.003 mg/ml), the first generation of PTX preparation, Taxol®,4 is formulated in mixed solvents of polyoxyethylated castor oil (Cremophor EL) and dehydrated ethanol (50/50, v/v).5 However, direct injection of Cremophor EL can cause serious side effects, such as allergic reactions, in about 30% of patients. To exclude the need for Cremophor EL, the second generation of PTX formulations were developed, such as Abraxane® (albumin-paclitaxel nanoparticles) and Genexol-PM® (poly(ethylene glycol)-block-poly(D,L-lactide) (mPEG-PDLLA) micelles).6,7 In addition, the polymeric pro-drug of PTX also has attracted wide attention for their enhanced water solubility, for instance, PEG-PTX8 and Xyotax™ (poly(L-glutamic acid)-paclitaxel (PGA-PTX) ),9 where the latter one has already advanced to the clinical trials. We previously have also developed a polymeric pro-drug, poly (L-γ-glutamyl-glutamine)-paclitaxel (PGG-PTX), which displayed a more increased solubility and antitumor effect than PGA-PTX.10 Compared with Taxol®, these new generations of PTX showed reduced side effects, improved tolerated dose and good pharmacokinetic properties. Despite this, there are still two problems with them: the short blood-circulating time and poor drug release profile in vivo. For example, the area under the curve (AUC) and plasma half-life of Genexol-PM remained well below that of Taxol in clinical studies.11 Xyotax™, due to the stable conjugation of PTX to the polymer backbone PGA, the exposure of bioactive drug to the patient can be quite low even when the total PTX dose becomes very high.12 Releasing drug timely and adequately in tumor sites is a critical step to exert the antitumor effect of nano drug delivery systems (NDDSs). For this purpose, researchers have made a mighty advance in the tumor microenvironment responsive NDDSs, including pH-sensitive,13 temperature-sensitive and enzyme-sensitive systems,2,14 which could improve the drug release behavior and drug efficacy.15 Although those polymeric pro-drugs of PTX showed higher solubility, better pharmacokinetic behavior and lower side effect than Taxol did, the inadequate drug release is still a common problem with them. For instance, the in vitro drug release of PGG-PTX NPs we previously prepared was only up to 10% within 60 hours. To solve this problem, based on the low pH in tumor tissue (pH 6 - 6.5) and lysosomes (< pH 5.0) of tumor cells, our group synthesized a pH-sensitive self-assembled polymeric pro-drug of PTX, PGSC-PTX NPs (Fig. 1). The cumulative release rate of PTX from PGSC-PTX NPs16 was significantly increased at pH ~ 6.5, showing good pH sensitivity. The long-circulating effect is another essential factor for successful tumor-targeting therapy with NDDSs. The most widely-used method is to modify the NPs surface with the highly hydrophilic polyethylene glycol (PEG), and there have been a large number of researches on the PEGylated NDDSs.17-19 However, several


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studies have found that the body can produce immune response to PEGylated NDDSs, which can be captured by reticuloendothelial system (RES).20 To solve this problem, Liangfang Zhang group developed a red blood cell membrane (RBCm) wrapped NPs, showing a good long-circulating effect.21-24 After wrapped by RBCm, the circulation half-life of NPs can be prolonged by nearly 2.5 fold than that of PEGylated NPs. Besides, the accessibility and low immunogenicity of the RBCm, can greatly increase the druggability of drug delivery system.25 Therefore, the RBCm-wrapping technology provides a simple and practical method for increasing the in vivo circulation time and improving the effectiveness and druggability of NDDSs. Hence, we here designed and prepared a RBCm-wrapped pH-sensitive polymeric pro-drug delivery system of PTX, PGSC-PTX@RBCm NPs, to increase the circulating time of PGSC-PTX NPs and improve PTX release at the acidic environment. This study was carried out to verify our hypothesis that, the red cell membrane can ensure the long circulation time of PGSC-PTX@RBCm NPs in the blood, and that PGSC-PTX NPs can be sensitively degraded in the acidic environment of the tumor tissue, and release a sufficient amount of PTX to kill tumor cells.

Results Preparation and characterization of PGSC-PTX@RBCm NPs In this study, to prolong the circulating time in vivo and release PTX timely in acidic environment, RBCm was wrapped to the surface of acidic sensitive polymeric pro-drug PGSC-PTX to prepare PGSC-PTX@RBCm NPs. The preparation process of PGSC-PTX@RBCm NPs included two steps: membrane vesicle derivation from RBCs and vesicle-particle fusion (Fig. 1). The first step was performed as a previously reported method with little modifications.26 Briefly, the RBCs separated from fresh blood were placed in a hypotonic environment to rupture member, and its intracellular contents were removed and collected vesicles. With an Avanti mini extruder, the resulting vesicles were subsequently extruded trough polycarbonate porous membranes with size of 400 nm, 200 nm and then 100 nm. The second step is the fusion of RBCm membrane vesicles with PGSC-PTX NPs. 8 mg of PGSC-PTX NPs was mixed with RBCm vesicles extracted from1 mL of whole blood and then extruded through a 100 nm polycarbonate porous membrane for 7 times to obtain the PGSC-PTX@RBCm NPs.



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Fig. 1. Schematic diagram of the preparation process of the PGSC-PTX@RBCm NPs. The particle sizes of PGSC-PTX NPs and PGSC-PTX@RBCm NPs were characterized by DLS method. These results showed that the uniform particle size of PGSC-PTX NPs and PGSC-PTX@RBCm NPs were about 50 nm (PDI = 0.069) (Fig. 2A) and 130 nm (PDI = 0.108) (Fig. 2B), respectively. In order to observe the morphology of NPs, the particles were subjected to negative staining with phosphotungstic acid and then observed by TEM (Fig. 2C, D). Both PGSC-PTX NPs and PGSC-PTX@RBCm NPs showed well-defined spherical morphology. More importantly, as shown in the TEM image of PGSC-PTX@RBCm NPs, an obvious layer was coated on the surface of NPs compared to PGSC-PTX NPs. The thickness of the layer is determined to be about 10 - 20 nm, which is in agreement with that of the RBCs membrane in the previous reports.26,27 In addition, SDS-PAGE protein analysis of PGSC-PTX@RBCm NPs and natural RBC membranes was performed in parallel, and the results (Fig. 2E) showed that the majority of endogenous membrane proteins are preserved on PGSC-PTX@RBCm NPs compared with the RBC membranes, which may allow the NPs to invade immune cognition. These results suggest that the RBCm have been successfully encapsulated on the PGSC-PTX NPs.

Fig. 2. Characterization of PGSC-PTX NPs and PGSC-PTX@RBCm NPs. (A) DLS analysis of PGSC-PTX NPs;(B) DLS analysis of PGSC-PTX@RBCm NPs; (C) TEM image of PGSC-PTX NPs; (D) TEM image of PGSC-PTX@RBCm NPs. PGSC-PTX and PGSC-PTX@RBCm NPs all displayed uniform spherical morphology, as shown in the TEM images; (E) SDS-PAGE protein analysis of RBCm and PGSC-PTX@RBCm NPs.


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EE and LC are two key parameters for assessing the ability of polymers to capture drug. The LC of PGSC-PTX NPs was measured to be 32.8% by HPLC. The EE of PGSC-PTX@RBCm NPs was 26.2%, which was calculated as the percentage of the NPs in the drug delivery system. The long-term stability of PGSC-PTX NPs and PGSC-PTX@RBCm NPs was characterized by the change of particle sizes in two weeks. The results (Fig. 3) showed that the particle sizes are no obvious changes, indicating that both types of NPs have good stability.

Fig. 3. Stability of PGSC-PTX NPs and PGSC-PTX@RBCm NPs in PBS solution over two weeks at 4 °C. The PTX release from PGSC-PTX NPs or PGSC-PTX@RBCm NPs was performed in PBS with pH 7.4 and pH 6.5 at 37 °C for 108 h (Fig. 4). PTX release from the both NPs at pH 6.5 was remarkably higher than those at pH 7.4, respectively. At 108 h, the cumulative release rate of PTX from PGSC-PTX NPs arrived 38%. The results showed that PTX released from PGSC-PTX NPs and PGSC-PTX@RBCm NPs have pH sensitivity, and that RBCm wrapping could reduce the drug release to a certain extent, but not affect the pH sensitivity.



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Fig. 4. The PTX release curves of PGSC-PTX NPs and PGSC-PTX@RBCm NPs at pH 6.5 and pH 7.4 at 37 °C (n = 3, bars represent means ± SD).

Cellular uptake in vitro The cellular uptake of the two NPs on NCI-H460 cells was determined by CLSM and flow cytometer. The results showed in Fig. 5, the percentages of DiO-positive cells for control, PGSC-PTX/DiO NPs and PGSC-PTX/DiO@RBCm NPs were 0.17%, 91.07% and 93.08%, and the mean fluorescent intensities were 5.09, 1602.87 and 929.6, respectively. This results indicated that the uptake of the two NPs by NCI-H460 cells could be remarkably reduced by RBCm wrapping.

Fig. 5. The representative images of uptake by NCI-H460 and flow cytometry for control (A), PGSC-PTX/ DiO NPs (B) and PGSC-PTX/DiO@RBCm NPs (C). The cellular uptake of PGSC-PTX/DiO@RBCm NPs on NCI-H460 cells was slightly reduced by RBCm wrapping. Blue: Hoechst 33342 stained cell nucleus. Green: DiO-labeled nanoparticles. Red: Dil stained Red blood cell membrane.

Cytotoxicity assays in vitro The viability of NCI-H460 cells was measured after incubation with Taxol, PGSC-PTX NPs and PGSC-PTX@RBCm NPs. As shown in Fig. 6, the IC50 value for Taxol, PGSC-PTX NPs and PGSC-PTX@RBCm NPs was 0.00374 µg/mL, 0.1906 µg/mL and 0.3768 µg/mL, respectively. The results showed that Taxol had the strongest cytotoxicity, and that PGSC-PTX@RBCm NPs displayed decreased cytotoxicity compared with PGSC-PTX NPs. The results should result from that RBCm wrapping hindered the cellular uptake of NPs by tumor cells as shown in Fig. 5.


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Fig. 6. The cytotoxicity of Taxol, PGSC-PTX NPs and PGSC-PTX@RBCm NPs on NCI-H460 cells. PGSC-PTX NPs and PGSC-PTX@RBCm NPs showed significantly lower growth inhibitory effect than Taxol.

Immunogenic responses The immunogenic response was evaluated by the uptake by Raw 264.7 macrophages for NPs. The results (Fig. 7), the mean fluorescent intensities for PGSC-PTX/DiO NPs and PGSC-PTX/DiO@RBCm NPs were 831.42 and 344.61, respectively. The results indicated that the cellular uptakes of NPs were significantly decreased by RBCm wrapping.

Fig. 7. The representative images of uptake by Raw 264.7 for PGSC-PTX/ DiO NPs and PGSC-PTX/DiO@RBCm NPs. The mean fluorescent intensity was significantly reduced after RBCm wrapping.

Hemolysis study To determine whether PGSC-PTX NPs and PGSC-PTX@RBCm NPs can induce membrane damage, the hemolysis study was proformed. The results showed that the surfactants like Tween 80 and Cremophor EL caused significant red blood cell



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damage at 2 mg/mL, whereas PGSC-PTX NPs and PGSC-PTX@RBCm NPs had no hemolytic activity even at 8 mg/mL (Fig. 8). These data suggested that PGSC-PTX NPs and PGSC-PTX@RBCm NPs would not cause anemic toxicity after systemic intravenous injection.

Fig. 8. Hemolysis of RBCs after incubation with PGSC-PTX@RBCm NPs, PGSC-PTX NPs, EL and Tween 80.

Circulation half-life In order to evaluate the circulation half-life, the pharmacokinetic experiment of PGSC-PTX NPs and PGSC-PTX@RBCm NPs was carried on SD rats. The results (Fig. 9) showed that the elimination of PGSC-PTX@RBCm NPs in vivo is significantly slower than PGSC-PTX NPs. Besides, the area under curve within 72 h (AUC0-72h) for PGSC-PTX NPs and PGSC-PTX@RBCm NPs is 850.2 and 2082, respectively. These results suggested circulation time of NPs could prolong significantly after RBCm encapsulation and further increase the possibility of NPs tumor accumulation.

Fig. 9. The pharmacokinetic curves of the NPs. Blood taken at various time points after DiD-loaded NPs were i.v. injected to SD rats and the fluorescence was measured at 670nm.

Antitumor activity in vivo The antitumor efficacy in vivo was carried out on NCI-H460 tumor-bearing mice.


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The results (Fig. 10) showed that PGSC-PTX@RBCm NPs displayed significantly stronger antitumor effect compared with PTX (P < 0.001), PGSC-PTX NPs (P < 0.001), and saline (P < 0.001). This results suggested that RBCm wrapping could enhance the antitumor effect of NPs, which may result from the pH-sensitive drug release and prolonged circulation time as we mentioned above. The body weight of animals was almost increased. This indicated our NDDSs has little cytotoxicity.

Fig. 10. In vivo treatment of NCI-H460 tumor-bearing mice. Tumor volume curves (A); Relative weights change (B).

Discussion Long circulation is the prerequisite of sustained and tumor-targeted delivery of nanomedicines. In recent years, however, PEGylation of NPs, the gold standard of long circulation, was reported to be able to cause immunological responses. Therefore, researchers had been trying to find a kind of low immunogenic materials to be used for long circulating NPs. Correspondingly, Zhang et al. developed a RBCm wrapping technology, by which the RBCm can be completely packaged on the surface of NPs so as to achieve the goal of long circulation.28,29 Possibly by reducing macrophage uptake, the results suggested that the circulating time of NPs in vivo could be prolonged after RBCm coated, and it was confirmed by our results (Fig. 7). The results may be due to the expression of CD47 on RBCm that can inhibit phagocytosis by interacting with signal regulatory protein alpha (SIRPα) expression on macrophages.30 Although RBCm wrapping can evade the macrophage phagocytosis, we also found an unfavorable result that it can decrease the NP uptake by tumor cells, further decreasing the in vitro cytotoxicity. According to the report,31 the size of NPs



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was found to have great influence on the cellular uptake. Thus, the increased particle size after RBCm coating may be another reason for the decreased cellular uptake. Therefore, how to evade the macrophage phagocytosis while not decreasing the uptake by tumor cells should be one of our future research issues. Active targeting mediated by targeting molecules like peptides or antibodies may be a good choice. Along with the in-depth study on tumor microenvironment, pH sensitive polymeric pro-drug have attracted more and more attention. PGSC-PTX we here used in this study contains a flexible carbocistein linker between PGA and PTX, which can break and trigger drug release in tumor acidic microenvironment and release PTX rapidly. Compared with PTX, the water solubility of PGSC-PTX was increased, and the side effects can be decreased because no organic solvent was used. PGSC-PTX can also self-assemble to form NPs, increased the accumulation of PTX in tumor site through the EPR effect. More importantly, PGSC-PTX NPs could timely release PTX in the acidic tumor environment to kill tumor cells. Our results showed that the amounts of PTX release at pH 7.4 are lower than those at pH 6.5 for both PGSC-PTX@RBCm NPs and PGSC-PTX NPs, which demonstrated the acidic sensitivity of the polymer. In addition, we found that the difference between drug release of PGSC-PTX@RBCm NPs at pH 6.5 and that at pH 7.4 was significantly less than PGSC-PTX NPs, suggesting the decrease of acidic sensitivity of PGSC-PTX after RBCm wrapping. This may result from that RBCm wrapping prevented the pH sensitive linker from contacting with the acidic tumor environment. This problem may be solved by increasing the acidic sensitivity of RBCm by inserting acid sensitive molecules into the phospholipid membrane. Besides, the pH effect on in vitro cellular uptake was investigated (Fig. S1 and Fig. S2). Unlike the “smart” pH-sensitive polymers,32 the pH sensitive linker in the PGSC-PTX between PGA and PTX, which almost not affect cellular uptake at pH 6.5 and pH 7.4. The pH change from 7.4 to 6.5 may have a very limited influence on the charge properties of the PDCs, and thereby on the cellular uptake. Similar results were also observed on the cellular uptake by Raw 264.7 cells. Tumor-targeted NDDS in vivo process is consisted of seven steps: blood circulation, tumor accumulation, tumor tissue penetration, target cells internalization, lysosome escape, drug release and drug response. A problem in any step will lead to the failure of the tumor treatment.33 Our NDDSs have just solved the problems in blood circulation and drug release. The problems in other steps remain to be solved by other techniques, such as tumor-penetrating-peptide modification and insertion of fusogenic lipid.

Conclusion In summary, PGSC-PTX@RBCm NPs exhibited a raised therapeutic effect on lung cancer, in view of their characteristics of pH sensitivity and long circulation, indicating that pH sensitive linker and RBCm wrapping are effective strategies for improving the antitumor effect of PTX. The research could provide a solution for the current predicament of tumor-targeted NDDSs.

Materials and methods ACS Paragon Plus Environment

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Materials Polymeric pro-drugs (PGSC-PTX) were synthesized by our laboratory. Taxol was purchased from Fudan University Shanghai Cancer Center. PTX was purchased from Aladdin. Cell Counting Kit-8 (CCK-8) was supplied by DOJINDO Laboratories chemical technology (Shanghai) co., LTD. 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO), 1,1'-dioctadecyl -3,3,3',3'- tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were supplied by Tianjin Biolite Biotech Co., LTD. Hoechst 33342 and DAPI were supplied from Beyotime Institute of Biotechnology. NCI-H460 cell line was supplied from Chinese Academy of Sciences. BABL/C nude mice were supplied by SLAC Ltd (Shanghai, China). All experiments associated with mice were performed by the guidelines of the Institutional Animal Care and Use Committee of East China Normal University.

Preparation of PGSC-PTX@RBCm NPs RBC ghost derivation RBC ghosts without cytoplasmic contents were prepared according to the previously reported protocols with modifications.34 In brief, the whole blood was first collected from male nude mice (6 - 8 weeks) and then centrifuged at 900 × g for 20 min at 4 °C. After removing the supernatant, the RBCs were collected. The obtained intact RBCs were washed with cold phosphate buffered saline (1 × PBS), suspended with 0.2 mM EDTA in an ice bath for 20 min to achieve hemolysis and then centrifuged at 21000 × g for 7 min. As a result, the pink pellet was obtained with the hemoglobin removed. Preparation of RBC-derived vesicles The collected RBCs ghosts were sonicated for 5 min using an FS30D bath sonicator (59 kHz, 100 W, Fisher Scientific). With an Avanti mini extruder (Avanti Polar Lipids), the resulting vesicles were subsequently extruded trough polycarbonate porous membranes with size of 400 nm, 200 nm and then 100 nm. Preparation of PGSC-PTX@RBCm NPs The preparation of PGSC-PTX@RBCm NPs was prepared according to the published procedure.35 In brief, 1 mg of PGSC-PTX NPs was added into RBCm-derived vesicles extracted from 1 mL of whole blood and then extruded through a 100 nm polycarbonate porous membrane for 7 times using a liposome mini extruder (Avanti). The resulting solution was performed of a gel column chromatography (G50) connecting AKTA purifier (GE, USA) to collect PGSC-PTX@RBCm NPs.

Characterization of PGSC-PTX NPs and PGSC-PTX@RBCm NPs



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Particle size determination Dynamic light scattering (DLS, Mastersizer2000) was used to measure the particle sizes of PGSC-PTX NPs and PGSC-PTX@RBCm NPs. The samples (n = 3) were measured at room temperature for 10 min. Morphology of PGSC-PTX NPs and PGSC-PTX@RBCm NPs Transmission electron microscopy (TEM) was used to image the morphology of PGSC-PTX NPs and PGSC-PTX@RBCm NPs. Negative staining was performed and the TEM test was performed with a JEM-2100 electron microscope (Hitachi, Tokyo, Japan). Encapsulation efficiency (EE) and loading capacity (LC) The drug payload of PGSC-PTX NPs was 32.8%, the PTX content was measured using a standard curve generated via a series of PTX solution of certain concentration by HPLC. The EE was assayed as the ratio of the drug amount in the NPs to the total drug amount added in the solution. The LC was calculated as the percentage of the drug amount incorporated in the NPs to the total amount of the NPs. Characterization of protein in RBCm and PGSC-PTX@RBCm NPs To validate the RBC membrane coating process, the proteins in RBC membranes before and after coating was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The RBC membranes and PGSC-PTX@RBCm NPs were prepared in SDS sample buffer (Beyotime) and heated at 100 °C for 10 min. The samples were then run on a well containing 10% SDS polyacrylamide gel (Beyotime) at 150 V for 1 h, then the obtained polyacrylamide gel was stained in Simply Blue and destained overnight before imaging. Long-term stability of PGSC-PTX NPs and PGSC-PTX@RBCm NPs NPs solution (2.0 mg/mL) was suspended in PBS and equilibrated for 5 min at room temperature. The particle sizes and the polydispersity index (PDI) of the two NPs were monitored by DLS. The test lasted two weeks. PTX release from NPs in vitro The purpose of this experiment is to verify the pH-dependent PTX release from PGSC-PTX NPs and PGSC-PTX@RBCm NPs. NPs were suspended in fresh PBS (pH 7.4 and pH 6.5) with a final concentration of 0.5 mg/mL and packed in 5 mL centrifuge tube. Then they were shaken at 120 r/min with an incubator shaker at 37 °C. At predetermined time points, the sample was taken out and PTX was extracted by 1 mL ethyl acetate. Then PTX was dissolved in acetonitrile after drying using a nitrogen blowing instrument. Finally, the drug concentration was determined by HPLC.

Cellular uptake in vitro Preparation of DiO-loaded NPs


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The emulsification-solvent evaporation method was used to prepare PGSC-PTX/DiO NPs.36 In brief, PGSC-PTX dissolved in sodium cholate solution was used as the water phase, then mixed with the organic phase prepared by DiO suspended in acetone and methylene chloride acetone (1:3, v/v). The mixture was emulsified in ice bath by ultrasonic method and evaporated with a rotary evaporator to remove the organic solvent. The obtained PGSC-PTX/DiO NPs were then purified by G50 gel column connecting AKTA purifier to remove the free DiO. After extrusion, 1 µL DiI (5 mM) was added to 1 mL PGSC-PTX/DiO@RBCm solution and incubated for 30 min at 37 °C to stain the RBCm coated on the surface. The free DiI were then removed by G50 gel column and stained PGSC-PTX/DiO@RBCm were collected. Cellular uptake by tumor cells Cellular uptake of the two NPs by NCI-H460 cells was test as follows. Cells were seeded in 2.5 cm dish, cultured overnight, and incubated with PGSC-PTX/DiO NPs and PGSC-PTX/DiO@RBCm NPs diluted in the FBS free culture medium at different pH values (pH 7.4 and pH 6.5) for 4 h. After that, cells were washed with PBS, fixed with 4% paraformaldehyde, stained with Hoechst33342, and observed with a confocal laser scanning microscope (CLSM). For quantitative flow cytometric analysis, the treated cells were trypsinized and centrifugated at 1000 rpm for 5 min. The cells at bottom were harvested, resuspended in 200 µL 1 × PBS, and analyzed by flow cytometry.

Cytotoxicity in vitro The cytotoxicity of the two NPs in vitro was determined using the CCK-8 assay.37 Cells were seeded in a 96-well plate (2000/well), cultured for 24 h, and treated with a series of dilutions of Taxol, PGSC-PTX NPs, and PGSC-PTX@RBCm NPs (100 µL/ well) for 48 h. After culturing with CCK-8 Solution (10 µL/ well) for 4 h, the absorbance was measured using a microplate reader (Thermo Multiskan MK3; Thermo Scientific, Waltham, MA) at 450 nm. The survival rate was calculated by the following formula: viability rate = [(ODtest group – ODBlank)/(ODcontrol group – ODBlank] × 100%. The IC50 was calculated by GraphPad Prism v7.00.

Immunogenic responses The immunogenic response was evaluated using the cellular uptake of the two NPs by Raw 264.7 macrophages in vitro. Cells were seeded in 24-well plates (3 × 104/well), cultured for 24 h, washed with PBS, and incubated with PGSC-PTX/DiO NPs and PGSC-PTX/DiO@RBCm NPs for 4 h. The quantitative analysis of cellular uptake was performed using flow cytometry as mentioned above.

Hemolysis study To assess the blood compatibility, the hemolysis assay was performed following the published procedure.38 Briefly, fresh blood collected from rats was washed with N.S. and centrifuged at 1500 rpm for 20 min. The collected RBCs suspension was diluted with N.S. to obtain a 2% suspension (v/v). PGSC-PTX NPs and



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PGSC-PTX@RBCm NPs, Tween 80, Cremophor EL, distilled water and N.S. were added into the RBC suspension and incubated at 37°C for 1 h, respectively. To remove non-lysed RBCs, the samples were centrifuged at 3000 rpm for 10 min. The spectrophotometric detection was used to analyze the hemoglobin content of the collected supernatants (n = 3) at 416 nm.

Circulation half-life To evaluate the circulation half-life of PGSC-PTX NPs and PGSC-PTX@RBCm NPs, the hydrophobic fluorescent dye DiD was encapsulated into the two NPs. For these two NPs, 1 mL of 5 mg/mL DiD-loaded NPs was injected into SD rats (n = 6) through tail-vein injection. 500 µL of blood were collected at determined time points following the injection. Plasma samples were collected from blood by centrifugation immediately at 4,000× g for 10 min and then detected by fluorescence spectrophotometer at 670 nm.39

Antitumor activity in vivo To obtain the tumor-bearing mice, 5 × 106 NCI-H460 cells were implanted into the male nude mice (6 - 8 weeks) subcutaneously. When the tumors reached a size of 100 mm3, the drug treatment was started. The mice were divided into four groups randomly: 1) N.S.; 2) Taxol (50 mg/kg); 3) PGSC-PTX NPs (80 mg/kg of PTX); 4) PGSC-PTX@RBCm NPs (80 mg/kg of PTX) (n = 6). The mice were i.v. injected via tail-vein every three days for a total of four times. The tumor volume and body weight of the mice were measured two or three times per week until the volume of the N.S. group reached 3000 mm3, at which time all the mice were terminated.

Statistical analysis Statistical analysis was performed with two-tailed student’s t-test, one-way ANOVA and two-way ANOVA. The differences were considered to be significant at P < 0.05 and very significant at P < 0.01.

Acknowledgments This work was supported by National Basic Research Program of China (2013CB932500, 2014CB744505), National Natural Science Foundation of China (60976004, 81430040, 81571738), “985” grants of East China Normal University (ECNU), Zhejiang Provincial Science and Technology Department Public Technology Application Research Program (2015C33285), and Zhejiang Provincial Natural Science Foundation of China (Y14H300002, LY15H180003). We wish to thank Yiwen Wang and Bing Ni for their assistance on TEM and CLSM test.

Conflict of interest statement The authors declare no competing ﬁnancial interest.

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Table of Contents graphic (TOC) 50x39mm (300 x 300 DPI)


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Fig. 1 170x73mm (300 x 300 DPI)



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Fig. 2 170x59mm (300 x 300 DPI)


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Fig. 3 92x67mm (300 x 300 DPI)



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Fig. 4 80x60mm (300 x 300 DPI)


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Fig. 5 84x42mm (300 x 300 DPI)



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Fig. 6 82x59mm (300 x 300 DPI)


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Fig. 7 92x81mm (300 x 300 DPI)



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Fig. 8 78x55mm (300 x 300 DPI)


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Fig. 9 55x38mm (600 x 600 DPI)



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Fig. 10 110x81mm (600 x 600 DPI)


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Fig. S1 107x70mm (600 x 600 DPI)



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Fig. S2 58x65mm (600 x 600 DPI)


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Erythrocyte Membrane-Wrapped pH Sensitive Polymeric

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