Improved tumor tissue penetration and tumor cell uptake achieved by delayed charge reversal nanoparticles

Acta Biomaterialia xxx (2017) xxx–xxx

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Improved tumor tissue penetration and tumor cell uptake achieved by delayed charge reversal nanoparticles Jingxin Gou a, Yuheng Liang a, Linlin Miao a, Wei Guo b, Yanhui Chao a, Haibing He a, Yu Zhang a, Jingyu Yang b, Chunfu Wu b, Tian Yin c, Yanjiao Wang a, Xing Tang a,⇑ a b c

Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenyang 110016 China Department of Pharmacology, School of Life Sciences and Bio-pharmaceuticals, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenyang 110016 China School of Functional Food and Wine, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenyang 110016 China

a r t i c l e

i n f o

Article history: Received 15 May 2017 Received in revised form 12 August 2017 Accepted 17 August 2017 Available online xxxx Keywords: Polyion complex Cabazitaxel Tumor penetration Nanoparticle Drug delivery Charge reversal

a b s t r a c t The high affinity of positively charged nanoparticles to biological interfaces makes them easily taken up by tumor cells but limits their tumor permeation due to non-specific electrostatic interactions. In this study, polyion complex coated nanoparticles with different charge reversal profiles were developed to study the influence of charge reversal profile on tumor penetration. The system was constructed by polyion complex coating using micelles composed of poly (lysine)-b-polycaprolactone (PLys-b-PCL) as the cationic core and poly (glutamic acid)-g- methoxyl poly (ethylene glycol) (PGlu-g-mPEG) as the anionic coating material. Manipulation of charge reversal profile was achieved by controlling the polymer chain entanglement and electrostatic interaction in the polyion complex layer through glutaraldehydeinduced shell-crosslinking. The delayed charge reversal nanoparticles (CTCL30) could maintain negatively charged in pH 6.5 PBS for at least 2 h and exhibit pH-responsive cytotoxicity and cellular uptake in an extended time scale. Compared with a faster charge reversal counterpart (CTCL70) with similar pharmacokinetic profile, CTCL30 showed deeper penetration, higher in vivo tumor cell uptake and stronger antitumor activity in vivo (tumor inhibition rate: 72.3% vs 60.2%, compared with CTCL70). These results indicate that the delayed charge reversal strategy could improve therapeutic effect via facilitating tumor penetration. Statement of Significance Here, the high tumor penetration capability of PEG-coated nanoparticles and the high cellular uptake of cationic nanoparticles were combined by a delayed charge reversal drug delivery system. This drug delivery system was composed of a drug-loading cationic inner core and a polyion complex coating. Manipulation of charge reversal profile was realized by varying the crosslinking degree of the shell of the cationic inner core, through which changed the strength of the polyion complex layer. Nanoparticles with delayed charge reversal profile exhibited improved tumor penetration, in vivo tumor cell uptake and in vivo tumor growth inhibition effect although they have similar pharmacokinetic and biodistribution behaviors with their instant charge reversal counterpart. Ó 2017 Published by Elsevier Ltd on behalf of Acta Materialia Inc.

1. Introduction Nanoscale drug delivery systems (NDDSs) are currently efficient tools in enhancing the therapeutic effect of chemotherapeutic agents due to improved pharmacokinetic profile as well as cellular

⇑ Corresponding author. E-mail address: [email protected] (X. Tang).

uptake [1–5], and most importantly, preferred accumulation in solid tumors through the enhanced permeation and retention (EPR) effect [6–8]. However, due to the high interstitial pressure and dense extracellular matrix of the tumor tissue [9–11], most of the nanoparticles were restricted in the vicinity of tumor vasculatures after extravasation, resulting in limited therapeutic effect of NDDSs due to inadequate drug exposure over tumor cells. So, efficient tumor penetration has been recognized as another key factor influencing the antitumor performance of NDDSs.

http://dx.doi.org/10.1016/j.actbio.2017.08.025 1742-7061/Ó 2017 Published by Elsevier Ltd on behalf of Acta Materialia Inc.

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Surface charge is a key parameter determining the performance of nanoparticles: positively charged nanoparticles could be preferentially taken up by cells via ‘‘electrostatic attraction mediated targeting” [12–14], however, this strong adhesion between cationic nanoparticles and bio-interfaces is a doubleedged sword which could indeed improve cellular uptake but, in the meantime, severely limit the amount of drug reaching tumor site due to shortened blood circulation [15–17]. Moreover, positively charged nanoparticles would primarily be ‘‘arrested” by the cells resided in the periphery of tumor vessels, thus limit their intratumoral distribution [18]. In contrast, neutral or negatively charged nanoparticles could travel longer distances in tumor tissue than the positively charged ones [19,20]. So, to utilize the advantaged cell internalization and avoid the limited tumor penetration of positively charged nanoparticles both induced by non-specific electrostatic interaction, it is hypothesized that NDDSs with delayed charge reversal profile might be capable of promoting tumor penetration without affecting cell internalization. Herein, nanoparticles with different charge reversal profiles were developed based on the platform of nanoparticles with polyion complex (PIC) coatings which showed negligible charge reversal behavior [21]. Manipulation of charge reversal profiles was realized by varying the shell crosslinking degree of cationic micelles self-assembled from poly (lysine)-b-polycaprolactone (PLys-b-PCL) using glutaraldehyde as the crosslinking agent, prior to electrostatic coating with poly (L-glutamic acid)-g-methoxyl poly (ethylene glycol) (PGlu-g-mPEG) (Scheme 1). Charge reversal caused by decoating of PGlu-g-mPEG under lowered pH was studied by monitoring the changes in n-potential and determination of the amount of remained PGlu-g-mPEG by GPC. The in vivo pharmacokinetics, biodistribution, intratumoral penetration and in vivo anti-tumor efficacy of nanoparticles with different charge reversal profiles were compared, and it was found that delayed charge reversal nanoparticles performed better in tumor penetration and tumor-growth inhibition.

2. Experimental section 2.1. Materials Polycaprolactone and poly (L-glutamic acid)-g-methoxyl poly (ethylene glycol) (PGlu-g-mPEG, dpGlu: 140, grafting ratiomPEG: 2%) were synthesized according to our previous work [22,23]. Ne-carbobenzyloxy-L-lysine was purchased from Enlai biological technology Co., Ltd. (Chengdu, China). Triphosgene was purchased from GL Biochem (Shanghai, China). Medium chain triglyceride (MCT) was provided by Lipoid KG (Ludwigshafen Germany). Cabazitaxel (CTX) was provided by the medicinal chemistry lab of Yantai University (Yantai, China). Coumarin 6 and rhodamine B isothiocyanate was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). 40 , 6-diamidino-2-phenylindole (DAPI) was purchased from Gen-view Scientific Inc. (USA). Fluorescein isothiocyanate (FITC)labeled Lycopersicon esculentum lectin were supplied by SigmaAldrich (St. Louis, MO, USA). All other reagents used were of analytical grade. 2.2. Cell lines and animals The human non-small cell lung cancer A549 cell line and mouse liver cancer H22 cell line were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Sprague-Dawley rats (male, body weight: 200 g ± 20 g) and kunming mice (male, body weight: 20 g ± 2 g) were provided by Liaoning Changsheng Biotech Co., Ltd. (Benxi, China). All animal experiments were approved by the Animal Ethics Committee of Shenyang Pharmaceutical University. 2.3. Preparation and characterization of nanoparticles Nanoparticles with different charge reversal profiles were prepared using cationic micelles as inner cores that were crosslinked

Scheme 1. Schematic illustration of preparation and tumor infiltration of nanoparticles with different charge reversal profiles. Charge reversal was manipulated by controlling the shell crosslinking degree: a low crosslinking degree indicated slower decoating due to higher chain entanglement in PIC layer and more –NH+3 available for electrostatic interaction (a); while reduced chain entanglement as well as available amount of –NH+3 resulted from increased crosslinking degree indicated faster decoating (b). After vessel extravasation, CTCL30 remained coated and gradually reversed surface charge during tumor penetration, this guarantees deeper penetration and higher cellular uptake; while the premature exposure of positive charge limited intratumoral distribution of CTCL70.

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in shells at different degrees (CL0, CL30 and CL70, CL indicates crosslink, the numbers indicate crosslinking degree), then the crosslinked micelles were coated with PGlu-g-mPEG to give coated crosslinked micelles (CTCL0, CTCL30 and CTCL70, CT indicates coated). Detailed preparation procedures were presented in supplementary materials. The particle size and n-potential of prepared micelles and coated nanoparticles with different crosslinking degrees were determined by dynamic light scattering (DLS) using a NICOMPTM 380 Submicron Particle Sizer (Particle Sizing System, Santa Barbara, CA) at room temperature. The morphology of coated nanoparticles and non-coated nanoparticles were examined by transmission electronic microscopy (JEM-2100, JEOL) with an acceleration voltage of 200 kV. Drug loading contents (DLCs) of coated nanoparticles were determined by HPLC method reported previously [22]. 2.4. Charge reversal study Charge reversal of CTCL0, CTCL30 and CTCL70 was studied by incubating the nanoparticles in pH 6.5 PBS. The changes in npotential and the amount of coated PGlu-g-mPEG were determined by DLS and gel permeation chromatograph (GPC), respectively. Detailed experimental procedures were presented in supplementary materials. 2.5. In vitro stability assays The colloidal stability and drug retention capability of coated nanoparticles in serum was studied by monitoring the variations in particle size and turbidity, as well as changes in the content of disulfiram (DSF). Detailed experimental procedures were presented in supplementary materials. 2.6. Cellular uptake study 2.6.1. Flow cytometry study A549 cells were seeded on 6-well plate at a density of 1  106 cells per well and incubated under 37 °C for 12 h to allow cell attachment. Then the culture medium was replaced with fresh medium containing coumarin 6-loaded CTCL30/CTCL70 and incubated for 2 or 4 h. After incubation, the cells were washed three times with cold PBS before being trypsinized and suspended in PBS. The fluorescence intensity of cells was measured using a flow cytometer (FACS Calibur, BD Biosciences). 2.6.2. Confocal laser scanning microscopy The influence of pH on cellular uptake of nanoparticles was studied using coumarin 6-loaded CTCL30 and CTCL70. A549 cells were seeded at a density of 1  104 cells per well in 0.5 mL dulbecco modified eagle medium (DMEM) culture medium on cover slips in 24-well plate and cultured overnight. Then the culture medium was replaced with fresh medium containing coumarin 6-loaded CTCL30/CTCL70 prior to 2 or 4 h incubation. Then the cells were washed 3 times with cold PBS and fixed by 4% paraformaldehyde at room temperature for 10 min. The cells were imaged by a confocal laser scanning microscope (CLSM) (Zeiss, LSM 710, Germany) after staining nucleus by DAPI. 2.7. Pharmacokinetic study Male Sprague-Dawley rats were randomly divided into 3 groups with 5 rats per group. CTX solution, CTX-loaded CTCL30 and CTCL70 were administered intravenously at a dose of 5 mg/kg. 0.5 mL blood was collected at predetermined time points (0.0833, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h), and plasma samples were

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obtained by centrifuging the collected blood samples at 4000 rpm for 10 min. Processing of plasma samples and determination of CTX concentration were conducted according to the method reported previously [22]. Pharmacokinetic parameters were calculated using the DAS 2.0 software (Drug and Statistics 2.0, Mathematical Pharmacology Professional Committee of China). 2.8. Biodistribution Male kunming mice bearing H22 xenograft tumors (tumor volume: around 200 mm3) were randomly divided into 9 groups (3 mice per group). CTX solution, CTX-loaded CTCL30 and CTCL70 were administered through tail vein at a dose of 10 mg/kg. 0.5, 4 and 8 h after administration, the mice was sacrificed to collect major organs (heart, liver, spleen, lung and kidney) and tumors. The collected tissues were washed with normal saline and dried with filter paper. Then the tissues were weight and cut into small pieces followed by homogenization, and CTX contents in the homogenates were determined by an ultra-performance liquid chromatography-tandem mass spectrometer (UPLC/MS/MS) (Waters Corp., USA). 2.9. In vivo tumor penetration and tumor cell uptake Rhodamine B isothiocyanate-labeled nanoparticles (CTCL30/ Rho and CTCL70/Rho) were injected via tail vein into H22 xenograft tumor-bearing mice. 4 h after administration, FITC-labeled Lycopersicon esculentum lectin (2 mg/kg) was intravenously administered 10 min prior to mouse sacrificing. The excised tumor was fixed in 4% paraformaldehyde for 12 h, and then immersed in 30% glucose solution for another 12 h. The tumor samples were sectioned into slices 20 lm in thickness, and then the slices were observed by CLSM. Tumor cell uptake of administered nanoparticles (CTCL30/Rho and CTCL70/Rho) was studied using flow cytometry: 4 h after injection, the tumors were excised, washed with cold normal saline, dried on filter paper and cut into small pieces. All the pieces were collected and digested with 0.1% collagenase for 6 h under 37 °C. Then, the cells were washed with cold PBS and passed through a 400-mesh sieve, counted and analyzed by a flow cytometer. Cells were recognized as ‘‘Rho-positive” when the fluorescence intensity exceeding 102. 2.10. In vivo antitumor study Male Kunming mice were randomly divided into 4 groups with 6 mice per group. Tumor bearing mice were established by injecting 1  106 cells subcutaneously in the right flank of mice. CTX solution, CTX-loaded CTCL30 and CTCL75 were injected intravenously via the tail vein at a dose of 10 mg/kg. Normal saline was administered in the same way as negative control (5 mL/kg). The formulations were first given when the sizes of tumors reached 150 mm3 (tumor volume = ab2/2, a and b are the largest and smallest diameter of the tumor, measured by a vernier caliper), then mice in different groups were further administered on the 3rd, 6th, and 9th day after first administration. Tumor inhibition rate (TIR,%) was calculated using the following equation: TIR (%) = (VcontrolVsample)/Vcontrol  100%, where Vsample and Vcontrol are the final and tumor volume of the treatment group and normal saline group. Five days after the last injection, the mice were sacrificed and the tumors were collected for TdT-mediated dUTP nickend labeling (TUNEL) and hematoxylin-eosin (H&E) staining. The sections were visual inspected after TUNEL staining to calculate apoptotic rates of different formulations, and detailed methods were included in the supplementary materials.

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2.11. Statistical analysis Statistical comparisons were conducted using Student’s t-test. Statistical significance was considered when p value <0.05. 3. Results and discussion 3.1. Preparation and characterization of nanoparticles The coated nanoparticles were prepared by a three-step approach based on a cationic core self-assembled from PLys-bPCL. MCT (10% of the weight of PLys-b-PCL) was introduced to improve the drug loading of the micelles [22]. The hybrid micelles were then shell-crosslinked. Upon shell-crosslinking, the particle size and n-potential showed a crosslinking degree-dependent reduction, probably due to less-stretched PLys chains and reduced cation amounts induced by imine formation between the –NH2 and glutaraldehyde in micelle shell (Table S2). Then, polyion complex (PIC) coating was conducted by dropping hybrid micelles with different crosslinking degrees (CL0, CL30 and CL70) into PGlu-g-mPEG solutions (Scheme 1). After coating, the particle size of coated nanoparticles showed an approximately 20 nm increase compared with the non-coated ones (Table S2), indicating the formation of PIC layer. The TEM images of CL0 (or cationic micelles), CTCL0, CTCL30 and CTCL70 were shown in Fig. 1.

constant during incubation, indicating that it was non-charge reversible; the n-potential of CTCL70 turned positive after 1 h incubation, while it took one more hour for CTCL30 to accomplish charge reversal (Fig. 2A). However, no changes in n-potential were observed when incubating the three nanoparticles in pH 7.4 PBS for 24 h (Fig. S2). These results indicated that the lowered environmental pH was responsible for charge reversal and control of charge reversal profile could be achieved by varying the degree of crosslinking. Then the amount of PGlu-g-mPEG remain coated during incubation was determined using GPC. The samples were acidified before lyophilization to facilitate dissolution of PGlu-gmPEG in DMF and, in the meantime, further reducing solubility of PLys-b-PCL in DMF by ionization of the PLys block. After 6 h incubation, the percentages of coating material remained on CTCL30 and CTCL70 were 34.7% ± 0.77% and 8.57% ± 6.1%, respectively; while more than 70% of PGlu-g-mPEG was found remain coated on CTCL0. This correlated well with the changes in npotential. The above results indicated that i) changing the shellcrosslinking degree of inner nanoparticle is a facile way realizing controllable charge reversal of PIC coated nanoparticles and ii) the charge reversal of coated nanoparticles was achieved by disassociation of the coating material from the inner core particle.

3.2. Charge reversal of coated nanoparticles with different crosslinking degrees The charge reversal of coated nanoparticles with different crosslinking degrees (CTCL0, CTCL30 and CTCL70) were first studied by monitoring the changes in n-potential of nanoparticles incubated in PBS with different pH values (7.4 and 6.5) [17,23–25]. After 6 h incubation in pH 6.5 PBS, it was found that the chargereversal profile of coated nanoparticles was proportionally related to crosslinking degree: the n-potential of CTCL0 remained nearly

Fig. 1. TEM images of prepared nanoparticles (Scale bars: CTCL30: 200 nm; others: 500 nm).

Fig. 2. Investigation in the charge reversal profiles of nanoparticles. (A). Changes in n-potential of CTCL0, CTCL30 and CTCL70 during 6-h incubation in pH 6.5 PBS; (B). Percentage of PGlu-g-mPEG remained as ‘‘coated” state in CTCL0, CTCL30 and CTCL70 during 6-h incubation in pH 6.5 PBS. (n = 3, mean ± SD).

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Currently, methods employed in the construction of NDDSs with prompt charge reversal profiles includes decoration of nanoparticles with functional groups respond sharply to tumor environmental pH [25–27] or shielding the cationic moieties with acid-labile bonds [28]. However, these methods, which relied on the changes in specific functional groups or chemical bonds, showed limited feasibility in achieving delayed charge reversal. It was found that nanoparticles with conventional PIC coating were incapable of charge reversal in acidic environment like tumor matrix [21]. However, methods like changing the ionization state of the coating materials by chemical modifications [17,21] or reducing chain entanglement within PIC layer by replacing polycations with small-molecular cationic surfactants [23,24], could endow PIC coated nanoparticles with pH-responsive charge reversal. Here, the shell of cationic micelles composed of PLys-b-PCL was crosslinked to control the amount and density of positive charge on surface of the inner positive particle, hence adjusting the strength of the PIC layer. Besides, introduction of glutaraldehyde helped the formation of a crosslinking network between PLys chains in the shell of the inner cationic particle, thus reducing chain entanglement between PLys and PGlu-g-mPEG. This would further reduce the strength of the PIC layer to facilitate charge reversal.

as it is a compound extremely instable in plasma [30] and relatively incompatible with PCL matrix [22], which means that the loaded DSF tended to migrate out of the nanoparticles and only the portion of DSF remain loaded could be determined by HPLC when incubating DSF-loaded nanoparticles (drug loading content: 1.1 ± 0.2%) in FBS (due to that cationic micelles could induce plasma clotting [20] and hence affect determination of DSF content, FBS was used instead of plasma in this study). Non-coated cationic micelles exhibited a poor cargo-holding capability as approximately 80% of loaded DSF leaked and degraded in the first 5 min of incubation, and the percentage of DSF remained after 2 h incubation was 4.58% ± 3.17%; while the coated nanoparticles (CTCL30 and CTCL70) performed better in resisting leakage with 27.3% ± 5.20% and 18.9% ± 1.54% of DSF remained, respectively, after 2 h incubation (Fig. 3C). Moreover, the results of in vitro release study also demonstrated that PIC coating was helpful in sustaining drug release [31] (Fig. S3). From these results it could be concluded that the existence of PIC coating is crucial in stabilization of nanoparticles in biological environments [31–33].

3.3. In vitro stability of nanoparticles with different charge reversal profiles

Coumarin 6-loaded CTCL30 and CTCL70 (CTCL30/Cou and CTCL70/Cou) were incubated with A549 cells under three conditions (pH 7.4, 2 h; pH 6.5, 2 h; pH 6.5, 4 h) to investigate the influence of coated state as well as decoating rate on the cellular uptake of nanoparticles with different charge reversal profiles. The treated cells were analyzed with flow cytometry and CLSM. After 2 h incubation in pH 7.4 medium, cells treated with both nanoparticles were found with relatively lower coumarin 6 fluorescence (Fig. 4, A, D and G) due to the steric hindrance of PEG chains limited effective contact between cells and nanoparticles [27]. However, when incubating the cells in pH 6.5 medium for 4 h with CTCL30/Cou and CTCL70/Cou, a 3-fold higher intracellular fluorescent intensity was observed (Fig. 4, C, F and G). These results indicated that i) the exposure of cationic poly (lysine) shell could facilitate internalization of nanoparticles due to electrostatic attraction mediated targeting [12,13,34]; ii) a 4-h incubation in pH 6.5 medium was long enough for complete charge reversal of both CTCL30 and CTCL70. The role of charge reversal profile on cellular uptake was studied by shortening the incubation time to 2 h: compared with the cells treated with CTCL30/Cou for 2 h in pH 7.4 medium, the cellular uptake of CTCL30/Cou was minimally improved by lowering the environmental pH (Fig. 4, A, B and G); while when incubating the cells with CTCL70/Cou for a same period of time in pH 6.5 medium, the intracellular coumarin 6 fluorescence intensity was

Stability in biological environment is a primary concern of NDDSs. In this study, the PIC layer was impaired to realize charge reversal, so it is necessary to test the stability of the nanoparticles. The colloidal stability of the prepared nanoparticles in fetal bovine serum (FBS) was first studied in vitro by monitoring the variations in particle size and sample transmittance. Due to the protective effect of PEG chains on the surface of the nanoparticles, the coated nanoparticles (CTCL30 and CTCL70) were relatively stable in FBS during 24 h with particle sizes changed from 138.2 ± 2.2 nm to 149.8 ± 8.1 nm (CTCL30) and 127.3 ± 1.7 nm to 134.9 ± 3.0 nm (CTCL70), respectively. However, the particle size of the uncoated cationic micelles increased drastically from 131.2 ± 3.5 nm to 153.2 ± 7.3 nm (Fig. 3A), probably due to protein adsorption [20,29]. The variations in particle sizes were correlated with changes in sample transmittance, and the nearly unchanged transmittance of CTCL30 and CTCL70 indicated good colloidal stability in FBS (Fig. 3B). Beside particle size, the capability of nanoparticles in holding the loaded cargo is another pre-requisite in guaranteeing effective drug delivery. In this study, disulfiram (DSF) was used as a tool drug to study cargo-holding capability of prepared nanoparticles

3.4. Cellular uptake and cytotoxicity of nanoparticles with different charge reversal profiles

Fig. 3. In vitro stability studies of nanoparticles. (A). Variations in particle size of cationic micelles, CTCL30 and CTCL70 incubated in FBS; (B). Variations in transmittance of cationic micelles, CTCL30 and CTCL70 incubated in FBS measured at 750 nm; and (C). Variations of disulfiram (DSF) contents in cationic micelles, CTCL30 and CTCL70 incubated in FBS (DSF was used as a tool drug examining the cargo-holding capability of nanoparticles due to its instability). (n = 3, mean ± SD).

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Fig. 4. In vitro cellular uptake studies of nanoparticles with different charge reversal profiles. (A) to (C), or (D) to (F): CLSM images of A549 cells incubated with CTCL30/Cou or CTCL70/Cou in pH 7.4 and 6.5 media after 2 or 4 h incubation, respectively; cell nucleus was stained with DAPI (Scale bars: 50 lm); (G) Flow cytometry analysis of A549 cells incubated with CTCL30/Cou (upper) and CTCL70/Cou (lower).

comparable with the cells incubated for 4 h (Fig. 4E, F and G). From these results, it was found that the decoated nanoparticles could be readily taken up by cells but the coated nanoparticles could not. Cellular uptake of a certain nanoparticle could be varied with the cell types used, but for a single type of cell, the cellular uptake improved with increased positive surface charge when keeping particle size constant [35], indicating the balance of surface charge could be a critical factor determining the cellular uptake of drug carriers. In summary, from the time-dependent cellular uptake of CTCL30 in acidic medium, it could be deduced that CTCL30 would remain low interactive with cells after first tumor vessel extravasation for a period of time. Cytotoxicity of empty and CTX-loaded formulations was studied in A549 and H22 cells by MTT assay. Both cell lines exhibited low cell viability when incubated with blank cationic micelles at 1 mg/mL due to the cytotoxicity of poly (lysine) [31], while the coated nanoparticles showed low cytotoxicity at equal PLys-bPCL concentration since the poly cations were shielded (Fig. S4). All formulations, except blank cationic micelles and CTX solution in DMSO, were found with improved cytotoxicity after preincubation in pH 6.5 buffer due to improved cellular uptake. And the improved cytotoxicity of the blank and CTX-loaded samples was the consequence of enhanced cellular uptake induced by re-exposure of poly (lysine) moiety. It should be noted that the cytotoxicity of CTX-loaded samples was believed to be induced primarily by CTX as the highest PLys-b-PCL concentration in the wells did not exceed 100 lg/mL. 3.5. Pharmacokinetics and biodistribution CTX solution, CTX-loaded CTCL30 and CTCL70 were administered to male Sprague-Dawley rats through tail vein at a dose of 5 mg/kg to evaluate whether the pharmacokinetic behaviors of coated nanoparticles were affected by their differences in charge reversal profiles, since the circulation time of nanoparticles would be shortened by the appearance of positive charge on the surface of nanoparticles [20]. According to the nearly superposed concentration-time curve of CTCL30 and CTCL70 it could be concluded that the pharmacokinetic behavior of the studied nanopar-

Fig. 5. Pharmacokinetic profiles of CTX solution, CTX-loaded CTCL30 and CTCL70 (n = 5, mean ± SD).

ticles was minimally affected by charge reversal profiles (Fig. 5). Combining the concentration-time curve and key pharmacokinetic parameters it was found that the two coated nanoparticles showed improved circulation profiles compared with CTX solution as reflected by prolonged half-life and reduced clearance (Fig. 5, Table 1). Biodistribution of CTX solution, CTCL30 and CTCL70 was compared in H22 xenograft tumor-bearing mice by determining CTX content in tissue 0.5, 4 and 8 h after intravenous injection using UPLC/MS/MS. CTX delivered by nanoparticles was found with higher accumulation in spleen and tumor than CTX solution, while the distribution of CTX in other organs showed no dependence on vehicle (Fig. 6), indicating that the nanoparticles are preferentially cleared by spleen, instead of liver. It has been reported that CTX is mainly metabolized in liver, with more than 80% of the drug metabolized by the CYP3A enzymes [36,37], so a lower distribution in liver would be helpful in maintaining the amount of CTX in the

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J. Gou et al. / Acta Biomaterialia xxx (2017) xxx–xxx Table 1 Pharmacokinetic parameters of CTX solution and CTX-loaded CTCL30 and CTCL70 after tail vein injection (n = 5, mean ± SD). Sample

AUC0t (lg/L*h)

t1/2 (h)

CL (L/h/kg)

MRT(0t) (h)

CTX solution CTCL30 CTCL70

1217.1 ± 427.35 3271.7 ± 244.06 2916.0 ± 209.77

0.562 ± 0.129 3.89 ± 1.84 3.54 ± 1.63

2.31 ± 0.898 1.43 ± 0.0941 1.23 ± 0.0976

0.419 ± 0.0308 1.04 ± 0.0832 0.965 ± 0.198

Fig. 6. Biodistribution of CTX in major organs 0.5 h (A), 4 h (B) and 8 h (C) after administration (n = 3, mean ± SD).

body. In CTCL30 and CTCL70 groups, different from the changes of CTX content in other organs, the tumor CTX contents remained relatively constant at each time point (Fig. 6). This phenomenon should first be ascribed to the EPR effect that facilitated tumor accumulation and in the meantime reduced nanoparticle clearance from tumor tissue due to absent lymphatic drainage. Besides, nanoparticles resided in tumor tissue would expose positive charge due to their charge reversal behavior, thus resist other means of clearance by interacting with negatively charged tumor tissue components. At each time point, the average tumor CTX content of CTCL30 group was higher than that of the CTCL70 group, but the difference was statistical insignificant. The comparable tumor accumulation between CTCL30 and CTCL70 was considered to be the consequence of their comparable particle size, surface property and pharmacokinetic profiles that made them extravasated in similar manners [9]. 3.6. In vivo tumor penetration and tumor cell uptake studies After vessel extravasation, CTCL30 and CTCL70 would exhibit different surface properties due to their different charge reversal profiles. And the influence of surface property on the tumor pene-

tration of nanoparticles was visualized using CLSM by administering rhodamine B isothiocyanate-labeled nanoparticles (CTCL30/ Rho and CTCL70/Rho). Tumor blood vessels were labeled with FITC-labeled Lycopersicon esculentum lectin [3]. H22 xenograft tumors were excised and cryo-sectioned 4 h after nanoparticle administration. The widespread red fluorescence in the field indicated better tumor penetration of CTCL30 (Fig. 7A). However, for the mouse treated by CTCL70, which has faster charge reversal rate, the red fluorescence located mainly in the peri-vascular region, indicating limited tumor penetration (Fig. 7B). Then, to test the effect of charge reversal profile on the intratumoral cellular uptake, CTCL30/Rho and CTCL70/Rho were i.v. injected to tumorbearing mice, and the intracellular RBIC intensity was measured by FCM after digesting the tumors into single cells. Cells were recognized as internalization ‘‘positive” when the RBIC fluorescent intensity exceeded 102. It was found that more than 80% of the tumors cells were positive when administering CTCL30/Rho, while the percentage of positive tumor cells in the mouse administered with CTCL70 was 23.3% (Fig. 7C). This higher in vivo tumor cell uptake of CTCL30 over CTCL70 was believed to be the consequence of delayed charge reversal of CTCL30, which increased the amount of cells exposed to the nanoparticles by the wider intratumoral

Fig. 7. Intratumoral distribution and tumor cell uptake of nanoparticles with different charge reversal profiles. Representative CLSM images of tumor cryo-sections (20 lm in thickness) obtained from H22 tumor-bearing mice administered with CTCL30/Rho (A) and CTCL70/Rho (B). Blood vessels in tumor were marked by FITC-labeled Lycopersicon esculentum lectin (green); Scale bars: 100 lm. (C). Flow cytometry analysis of cells harvested from H22 xenograft tumors 4 h after i.v. injection of CTCL30/Rho and CTCL70/ Rho. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 8. In vivo antitumor study of CTCL30 and CTCL70 in H22 xenograft tumor. (A). Tumor growth curve of mice receiving different treatments with drug administration indicated by black arrows; (B). Mice body weight variations during treatment; (C). Weight of dissected tumors after treatment; (* indicates significant difference, p < 0.05; n = 5, mean ± SD) (D). Photograph of tumors after treatment. (E). H&E and TUNEL staining of tumor sections treated with different formulations (Magnification: 400, scale bars: 100 lm).

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nanoparticle distribution as well as improved nanoparticle-cell interaction due to tumor acidity induced time-dependent charge reversal (Fig. 4). These results demonstrated that controlling the pH-responsive charge reversal profiles of nanoparticles could be a feasible way to facilitate nanoparticle migration in tumor matrix and cell internalization. 3.7. In vivo antitumor study The in vivo antitumor activity of CTCL30 and CTCL70 was studied in Kunming mice bearing H22 xenografts when the tumor sizes reached to approximately 150 mm3. The mice were administered with the formulations at 10 mg/kg via tail vein every 3 days with 4 injections in total. CTX solution exhibited moderate tumor inhibition compared with the rapid tumor growth in the negative control group (Fig. 8A), while the drastic fluctuation in mice body weights indicated the systemic toxicity induced by i.v. administration of CTX solution (Fig. 8B). Both the nanoparticles showed improved tumor inhibition compared with CTX solution as reflected by tumor inhibition rate (CTX solution: 39.3%; CTCL30: 72.3%; CTCL70: 60.2%; Fig. 8A to D) owing to improved tumor accumulation of CTX via EPR effect (Fig. 6). And the effect of delayed charge reversal on tumor inhibition was demonstrated by the stronger tumor inhibition effect of CTCL30 over CTCL70 (Fig. 8C) due to widespread intratumoral distribution as well as improved cellular uptake of CTCL30 (Fig. 8A and C). After TUNEL staining, it was found that CTX-loaded CTCL30 could generate more necrotic and apoptotic cells over other groups after i.v. injection (apoptotic rate: 62.25% ± 16.75% of CTCL30 versus 43.11% ± 11.26% of CTCL70), similar results were also found in H&E staining (Fig. 8E). Also, the evenly distributed apoptosis/necrosis regions in the H&E or TUNEL stained tumor sections treated by CTCL30 again indicated its improved tumor penetration capability, since segregated apoptosis/necrosis regions were observed in the tumor sections treated with low penetrative CTCL70 (Fig. 8E).

4. Conclusion The aim of this study was to find out the role of charge reversal profile on tumor penetration and tumor cell uptake. Firstly, a method controlling the charge reversal profiles of nanoparticles with PIC coatings was established, and then, nanoparticles (CTCL30 and CTCL70) with different charge reversal profiles in tumor microenvironment were prepared and were found with good colloidal stability and cargo-holding capability. The cellular uptake study revealed that the cellular uptake of both CTCL30 and CTCL70 were related with the presence of PGlu-g-mPEG coating. Both the CTX-loaded nanoparticles showed pH dependent cytotoxicity. In in vivo studies, CTCL30 and CTCL70 exhibited comparable pharmacokinetic profiles, and the intratumoral distribution study and in vivo tumor cell uptake study demonstrated that nanoparticles with delayed charge reversal profiles could simultaneously enhance tumor penetration and cell internalization. In H22 xenograft tumor bearing mice, CTCL30 exhibited superior tumor inhibition effect over CTCL70. In view of this, nanoparticles with delayed charge reversal profile displayed great potential as novel drug delivery systems that could improve tumor cell uptake without affecting tumor penetration. Acknowledgement This work was supported by Liaoning Excellent Talents in University (LJQ2014105) and China Postdoctoral Science Foundation (2016M600216).

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2017.08. 025.

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