Self-Assembled pH and Redox Dual Responsive

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Controlled Release and Delivery Systems

Self-assembled pH and redox dual responsive carboxymethylcellulosebased polymeric nanoparticles for efficient anticancer drugs co-delivery Kefeng Liu, Yanxue Liu, Chunxiao Li, Luying Wang, Jing Liu, and Jiandu Lei ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00920 • Publication Date (Web): 10 Oct 2018 Downloaded from http://pubs.acs.org on October 10, 2018

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ACS Biomaterials Science & Engineering

Self-assembled pH and redox dual responsive carboxymethylcellulose-based polymeric nanoparticles for efficient anticancer drugs codelivery Ke-Feng Liu,a Yan-Xue Liu,a Chun-Xiao Li,a Lu-Ying Wang,a Jing Liu,a Jian-Du Lei*a,b a

Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing 100083, P. R. China

b

Chemical and Biomolecular Engineering Department, University of California-Los Angeles, USA, CA 90095

Corresponding Authors *Email: [email protected] (Jian-Du Lei)

ACS Paragon Plus Environment

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ABSTRACT

To face the growing demand of polymeric nanoparticles with biocompatibility and drug release profile, in this work, a novel carboxymethylcellulose-based pH and redox dual-responsive polymeric nanoparticle, carboxymethyl cellulose-dithiopropionate hydrazide-8arm-polyethylene glycol-pterostilbene/10-hydroxy camptothecin (CTPP/HCPT) was prepared for efficient drugs co-delivery. These well-dispersed CTPP/HCPT NPs were prepared with the dimension of around 144 nm, exhibited high binary drug loading capacity and good biocompatibility. The most advantage of this design is that these nanoparticles can rapidly release the drug payload responding to intracellular acidic or reductive stimuli, while maintain sufficient stable in normal physiologic condition. The in vitro drug release study revealed that HCPT payload released from nanoparticles in a weakly acidic environment with 10 mM reductive glutathione was about 74.8%, which was 3.8-fold higher than at normal physiologic condition (~19.6%). Further in vitro and in vivo investigation demonstrated that such dual-responsive CTPP/HCPT NPs could potently kill cancer cell and suppress tumor growth with lower adverse effects. All these results suggested that CTPP/HCPT NPs were suitable as a potential and effective candidate for cancer therapy.

KEYWORDS: polymeric nanoparticles, biocompatibility, pH, redox, dual-responsive


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1. Introduction Self-assembled polymeric nanoparticles (NPs) have shown great potential in the field of drug delivery.1-4 This drug delivery system can effectively enhance therapy effect by improving water solubility and tolerance of drugs, enhancing accumulation at tumor site through the enhanced permeability and retention (EPR) effect, prolonging blood circulation time and decreasing adverse effects.5-6 Although effective, however, most current polymeric NPs are not satisfactory owing to uncontrolled drug release behavior and poor biocompatibility. In order to improve the drug release profile, intense efforts have been made to design and fabricate “intelligent” polymeric NPs, which could response to specific stimulus, such as redox potential, pH, light, temperature, and magnetic field.7-10 For instance, making use of acidic environments in cancerous tissues as compared to normal physiological condition, various pHresponsive NPs have been fabricated to specifically release drugs at the tumor acidic environments.11-14 Besides, various redox-responsive NPs have been produced to achieve specifically intracellular drug release by using the large difference in reduced glutathione (GSH) concentrations between the intracellular and the extracellular milieu.15-18 Although these single stimuli-responsive NPs exhibited improved drug release profile and enhanced therapeutic efficacy, however, multi-responsive NPs have been intensively pursued to further optimize drug release behavior and enhance anticancer effect.19-23 These multi-responsive NPs would provide unprecedented control over drug delivery and release, and thus leading to superior anticancer effect. In addition to controlled drug release profile, another indispensable request for polymeric NPs is good biocompatibility. To this end, various natural polymers, such as polysaccharide, protein, and polypeptide have been selected to fabricate NPs owe to their well-known good


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biocompatibility.24-29 Cellulose and its derivatives, as the most common and abundant polysaccharide,

also

provide many advantages,

such as

excellent

biocompatibility,

biodegradable, nontoxic, and easily chemical modification, thus making them potential candidate for drug delivery. Although the using of cellulose and its derivatives as carrier for drug delivery have been reported previously.30-32 However, carboxymethylcellulose-based pH and redox dualresponsive drug delivery system have been no reported to our best knowledge. Furthermore, a non-negligible drawback of current drug delivery system is that drug carriers are typically inert, and their sole role is to make the vehicles, which resulted in low drug loading capacity. In an amphiphilic polymer, the hydrophobic and hydrophilic segments are reached balanced to form nanoparticles. Actually, most anticancer drugs are hydrophobic. Therefore, we can use a hydrophobic drug to replace the hydrophobic part of amphiphilic polymer, which would minimize the use of other inert materials. Herein, a novel carboxymethylcellulose-based polymeric NP with pH and redox dualresponsiveness and good biocompatibility was prepared for efficient pterostilbene (PS) and 10Hydroxycamptothecin (HCPT) co-delivery (Figure 1). Notably, we artfully use hydrophobic anticancer drug PS as hydrophobic segment to replace the traditional inert material. PS is an antioxidant that is firstly discovered in red sandalwood. Recent studies find that pterostilbene can effectively inhibit cancer growth by altering the cell cycle (such as up-regulation of S phase cells), inhibiting of metastasis and inducing apoptosis.33 HCPT is an alkaloid anticancer agent, and exhibits potent antineoplastic effects in varied cancer cells by selectively inhibiting topoisomerase I (TopoI), preventing DNA replication and RNA synthesis. HCPT is specifically acts on cancer cells of S phase.34 Both HCPT and PS are naturally-derived anticancer drugs. Although exhibited potent anticancer effect, some drawbacks, such as poor water solubility,


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short blood circulation time and non-specific drug distribution, still restrict their further application in clinic. The delivery of PS and HCPT by CTPP/HCPT NPs not only overcome these drawbacks, but also could enhance the therapy effect of single anticancer drug through the synergistic effect. The in vitro drug release behaviors at different pH values with or without glutathione (GSH) were studied to verify the responsive release. Beside, the drug loading capacity, in vitro cytotoxicity, hemolysis study, cellular uptake and intracellular biodistribution were studied. Furthermore, the in vivo therapeutic efficiency and adverse effects were also evaluated on LLC-tumor bearing mice. 2. Materials and Methods 2.1 Materials Sodium carboxymethyl cellulose (CMC-Na, Mw = 120 KDa, DS = 0.82) was purchased from Sigma-Aldrich. 3,3'-dithiopropionate hydrazide (TPH) was purchased from J&K Scientific Co., Ltd (Beijing China). PS and HCPT were obtained from Aladin Co., Ltd., (Shanghai, China). 8arm-PEG-COOH (Mw = 5000 Da) was provided by JenKem Technology Co., Ltd (Beijing, China). 4-dimethylaminopyridine (DMAP), N-hydroxysuccinimide (NHS), and 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide hydrochloride (EDC•HCl) were purchased from SigmaAldrich. All the other reagents used in this work were received from Sigma-Aldrich. Mouse lewis lung cancer (LLC) cells and A549 cells were purchased from BeNa Culture Collection (Beijing, China). LLC cells and A549 cells were cultured in Dulbecco's modified eagle medium (DMEM) and Roswell Park Memorial Institute 1640 (RPMI-1640) medium containing 10% fetal bovine serum (FBS) and 1% antibiotics(penicillin and streptomycin), respectively.


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Mice (C57BL/6, female) were provided by Beijing HuaFuKang Biotechnology Co., Ltd (Beijing, China). Animal experiments in this work were performed according to the Guide for the Care and Use of Laboratory Animals. 2.2 Synthesis of CMC-TPH-PEG-PS (CTPP) Conjugates Synthesis of CMC-TPH: In brief, CMC-Na (1.0 g, 3.73 mmol COONa) was dissolved in 2-(NMorpholino) ethanesulfonic acid hydrate solution (MES, 100 mL, pH = 5.5), and activated with EDC (0.75 g, 3.73 mmol) and NHS (0.21 g, 1.87 mmol) for 15 min. Subsequently, TPH (1.78 g, 7.46 mmol) was added, and reacted for 24 h under gentle stirring. The mixture solution was then dialyzed (MWCO 3500 Da) against water for three times (3 × 4 h) to remove unreacted regents. Finally, CMC-TPH was obtained as a white porous sponge by lyophilization. Synthesis of 8arm-PEG-PS: 8arm-PEG-PS was synthesized according to our previously reported.35 Briefly, PS (0.25 g, 1.0 mmol) was first dissolved with dichloromethane (20 mL). 8arm-PEG-COOH (0.50 g, 0.1 mmol), DMAP (0.06 g, 0.5 mmol) and EDC (0.19 g, 1.0 mmol), were subsequently added, and reacted under gentle stirring overnight. The crude product was obtained by precipitated with excess ethyl ether, and then re-dissolved with deionized water, dialyzed against deionized water using a dialysis bag (MWCO 3500 Da) for three times (3 × 4 h). The final product 8arm-PEG-PS was obtained after lyophilized. Synthesis of CTPP: CTPP was synthesized through an amidation reaction. Briefly, excess 8arm-PEG-PS (0.35 g, 0.06 mmol) was dissolved in deionized water, and activated with EDC (27.5 mg, 0.15 mmol) and NHS (16.5 mg, 0.15 mmol) for 15 min. CMC-TPH (15 mg, 0.03 mmol) was subsequently added and reacted for 24 h under stirring. After that, the mixture was dialyzed (MWCO 10000 Da) against deionized water for 12 h. Finally, CTPP was obtained as a white powder by lyophilization.


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2.3 Preparation of CTPP and CTPP/HCPT NPs Nanoparticles were prepared via a simple nano-precipitation method.36 Free HCPT (0.20 g) and CTPP (0.50 g) were dissolved in 2 mL of dimethyl sulfoxide. Then the mixture solution was added dropwise to the beaker containing 18 mL of water. The CTPP/HCPT NPs solution were obtained after stirring for 15 min. The CTPP NPs were prepared through the same way. 2.4 Characterization of CTPP and CTPP/HCPT NPs The hydrodynamic diameter and surface charge of CTPP and CTPP/HCPT NPs were analyzed with dynamic light scattering (DLS, Malvern Zetasizer, Nano-ZS). The morphology of prepared NPs was observed with transmission electron microscope (TEM, JEM-1400, JEOL, Japan). The in vitro stability of NPs was analyzed using a simple storage method as described previously.37 The HCPT and PS content in NPs were measured using a high performance liquid chromatography (HPLC) (Waters 2695, USA). The drug loading capacity (DLC) and encapsulate efficiency (EE) were calculated as below: DLC (wt%) = weight of drug in nanoparticles/weight of nanoparticles × 100% EE (%) = weight of HCPT in nanoparticles/weight of HCPT added initially × 100% 2.5 In Vitro Drug Release Study To quantitatively assess the release rate of drugs, CTPP/HCPT NPs were dissolved in PBS buffer (pH 7.4) and acetate buffer (pH 5.0) with or without GSH (10 mM), and moved to dialysis bags with MWCO of 1000 Da. Subsequently, the dialysis bags were immersed into the same buffered solutions and subjected to shaking at 200 rpm at 37 °C. Periodically, release medium (1 mL) was withdraw and replaced with equal fresh buffer to keep the volume of release medium constant. The concentrations of HCPT and PS in medium were measured using a HPLC


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instrument (Waters 2695, USA) at 254 nm and 306 nm, respectively. HCPT and PS content were calculated using the standard curve of drugs in the respective buffers. 2.6 Hemolysis Study Fresh blood samples of healthy mice were collected and stirred with glass rod. Subsequently, appropriate amount of normal saline was added and centrifuged to separate the red blood cells (RBCs). The obtained RBCs were formulated 2% cell suspension with normal saline. For hemolysis study, RBCs suspension was separately treated different samples (CTPP NPs, CTPP/HCPT NPs, PEI, PBS or 1% Triton X-100) for 3 h at 37 °C. 1% Triton X-100 and PBS treatment groups were considered as positive and negative controls. Hemoglobin release was detected by a microplate spectrophotometer (Infinite M200, Tecan, Switzerland), and calculated as: Hemoglobin release (%) = (ODsamples – ODnegative)/(ODpositive– ODnegative) × 100%. 2.7 In Vitro Cytotoxicity Study The cytotoxicity of free drugs, CTPP and CTPP/HCPT NPs were measured by cell counting kit-8 (CCK-8) assay. Firstly, cells were seeded onto 96-well plates (5× 103 cells/well of LLC, 2× 104 cells/well of A549) and incubated with 180 µL culture medium for 24 h. Subsequently, the medium was replaced by different concentrations of drug formulations, and incubated for 72 h. At the end of the incubation, each sample solution was substituted with fresh medium (180 µL) and CCK-8 solution (20 µL), and incubated for another 4 h. The absorbance of different formulations was detected by a microplate reader (Infinite M200, Tecan, Switzerland). Cell viability was expressed as a percentage of absorbance of the control cells. The IC50 (the half maximal inhibitory concentration) values for each drug formulation was obtained by fitting the data using the Origins 8.6 (Origin Lab, Northampton, USA). 2.8 In Vitro Cellular Uptake Study


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The in vitro cellular uptake of CTPP/HCPT NPs was investigated by flow cytometry and confocal microscopy. Firstly, LLC cells (2×105 cells per well) were cultured onto a 6 well-plates and incubated overnight. Subsequently, culture medium was substituted with fresh medium containing free HCPT and freshly prepared CTPP/HCPT NPs (the concentration equivalent to free HCPT), and incubated for 4 h. For flow cytometer, cells were washed with DPBS several times, and measured using flow cytometer (Beckman Coulter, USA). For confocal images, cells were washed with cold DPBS several times, and then fixed with 4% formaldehyde, stained with DAPI (30 min). After washed several times with DPBS to remove excess DAPI, cell was observed using a confocal fluorescence microscope (CLSM) (Leica TCS SP8). 2.9 In Vivo Blood Circulation and Tissue Distribution LLC tumor-bearing mice were i.v. injected with different drug formulations: 1) free PS, 2) free HCPT, 3) CTPP NPs and CTPP/HCPT NPs. The dose of free PS was 20 mg/kg, CTPP NPs and CTPP/HCPT NPs doses were equal to free PS, and HCPT dose was equal to the HCPT content in CTPP/HCPT NPs. At selected time points, blood samples (~20 µL) were harvested from heart of mice, and the drug content in blood samples were measured by HPLC (Waters 2695, USA). To detect the in vivo biodistribution and organ clearance of CTPP/HCPT NPs, mice bearing LLC tumor were injected with CTPP/HCPT NPs (20 mg/kg) via tail vein and sacrificed at 4, 12 h, 24 h or 72 h. Major organs, such as heart, lung, kidney, liver, spleen and tumor were harvested, and HCPT and PS contents in tumors and organs were determined using HPLC. Drug content was described as µg drug/g tissue. 2.10 In Vivo Anticancer Study Tumor-bearing mice model was constructed by injecting LLC cells (1 × 107 per mouse) into the right flank of the mouse. Once the volume of tumor reached ~130 mm3, mice divided into


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five groups, and each group was treated with different formulations: 1) PBS, 2) free PS, 3) free HCPT, 4) CTPP NPs, and 5) CTPP/HCPT NPs via tail vein injection on day 0, 2, 4, 6 and 8. Mouse body weight and tumor volume were recorded every other day. In order to evaluate therapy efficiency, tumor volume (TV), relative tumor volume (RTV) and tumor growth inhibition (TGI) were calculated as follows: TV = L × W2/2, where L is the length of tumor, W is the width of tumor; RTV = Vt/V0, Vt is the tumor volume at treatment time, V0 is the tumor volume at the start of the experiment; TGI = (1-RTV of test group/RTV of control group) × 100%. 2.11 Detection of Adverse Effects Mice i.v. injected with different sample (PBS, free PS, free HCPT, CTPP and CTPP/HCPT NPs). Subsequently, cardiac blood samples were harvested to evaluate the adverse effects. Hypersensitivity reaction was reflected by the IgE level in blood. The hematologic toxicity was analyzed by detecting the number of white blood cell (WBC) and platelets (PLT) in blood using a hematology analyzer (DxH800, Beckman Coulter, USA). 3. Results and Discussion 3.1 Synthesis of CTPP Conjugates Synthesis of CMC-TPH: CMC-TPH was synthesized by an amidation reaction (Figure S1). The 1

H-NMR spectra of CMC, TPH, and CMC-TPH showed that the proton signals at 2.53 and 2.85

ppm (TPH) appeared in CMC-TPH, suggesting that TPH was successfully grafted onto the CMC chain (Figure S4). In addition, the partial methylene proton peak of TPH moved from 2.53 (1H, t) (blue 1) to 2.65 (1H, t) (red 1') owe to the formation of amido linkage. Synthesis of 8arm-PEG-PS: 8arm-PEG-PS conjugate was synthesized according to our previously reported (Figure S2). The 1H-NMR spectra showed that the proton signals from 6.41


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to 7.47 ppm (attributed to PS) appeared in 8arm-PEG-PS conjugate, suggesting the successful synthesis of 8arm-PEG-PS conjugate (Figure S5). The terminal methylene proton peak of PEG moved from 4.17 (1H, t) to 4.47 (1H, t) owe to the ester linkage formation. Importantly, a small terminal methylene proton peak of PEG remained 4.17 (1H, t) owe to the un-reacted carboxyl groups of PEG. Synthesis of CTPP: CTPP was synthesized by a simple amidation reaction (Figure S3). The 1

H-NMR of CTPP was shown in Figure S5, where chemical shift from 6.43 to 7.72 ppm belong

to PS, 3.26-4.51 ppm belong to PEG and CMC. The small terminal methylene proton peak of PEG moved from 4.17 (1H, t) to 4.41 (1H, t) owe to the formation of amido linkage between CMC-TPH and 8arm-PEG-PS. 3.2 Preparation and Characterization of CTPP/HCPT NPs As an amphiphilic polymer, CTPP can self-assemble into NPs in deionized water with hydrophilic CMC and PEG as the shell and the hydrophobic anticancer drug PS as the core. HCPT loaded into nanoparticles by a nano-precipitation method. The main physicochemical properties of prepared NPs were shown in Table 1. TEM images and size distribution of CTPP/HCPT NPs were determined and shown in Figure 2a-b. It can be seen that CTPP/HCPT NPs exhibited relatively uniform spherical shape with an average particle size about 144 nm. Additionally, little change was observed in particle size within 96 h storage, revealing its good in vitro stability (Figure 2c). 3.3 In Vitro Drug Release Profile To demonstrate the acid-mediated drug release of CTPP/HCPT NPs, drug release at different pH values (pH 7.4 and 5.0) was monitored to simulate the physiological condition and intracellular acidic environment, respectively. The release profiles showed that at pH 7.4 only


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19.4% PS released from CTPP/HCPT NPs within 72 h, while the release rate was remarkably facilitated at pH 5.0 with 63.5% of PS released within the same time (Figure 2d). The difference of PS release behaviors at different pH values was mainly attributed to that acid-labile ester linkage was cleave in acidic environment, while stable at neutral environment and retarded the PS release. The HCPT release behavior was also evaluated. As shown in Figure 2e, the release rate of HCPT from CTPP/HCPT NPs at pH 5.0 was faster than that of at pH 7.4. The cumulated release of HCPT at pH 5.0 was 52.7%, while at pH 7.4 was only 19.6%. The possible reason might be that the CTPP/HCPT NPs was disassembled at pH 5.0 owe to the release of hydrophobic PS, while it maintain stable at pH 7.4. The above data suggested that drug release behavior had significant pH-responsiveness. Moreover, the effect of reductive GSH on HCPT release was also evaluated. The results showed that the addition of GSH (10 mM) could significantly accelerated the rate of HCPT release both at pH 5.0 and pH 7.4, indicating that these nanoparticles were redox-sensitive (Figure 2e). 3.4 Hemolysis Study High hemolysis must be avoided for an injection drug formulation. The hemolysis study was conducted to demonstrate whether our nanoparticles could avoid intravascular or extravascular hemolysis (Figure 2f).The results showed that our nanoparticles resulted in significantly lower hemoglobin release (< 5%) compared with PEI under the same concentration, revealing the high safety. 3.5 In Vitro Cytotoxicity Study As shown in Figure 3a and b, all the drug formulations exhibited significantly dose-dependent toxicity in both LLC and A549 cells compared with control group. The highest toxicity was found in CTPP/HCPT NPs group, suggesting the enhanced anticancer activity. IC50 values of


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different drug formulations were evaluated and summarized in Table 2. Interestingly, the cytotoxicity of CTPP/HCPT NPs had significant time-dependence (Figure 3c and d). 3.6 Cellular Uptake Study The ability of CTPP/HCPT NPs to enter cancer cells was investigated by flow cytometry (Figure 4). The results showed that the mean fluorescence intensity in CTPP/HCPT NPs treatment group was about 4-fold higher than free HCPT treatment group, reflecting that the cellular uptake of HCPT was significantly enhanced by CTPP/HCPT NPs. The reason might be attributed to the efflux effect of p-glycoprotein toward hydrophobic free HCPT. However, nanoparticles could effectively avoid the effect of p-glycoprotein owe its internalized mainly by endocytic pathway.38 The enhanced cellular uptake of HCPT by nanoparticles was further confirmed by CLSM results. Strong fluorescence signals was observed in the cytoplasm and cell nucleus in CTPP/HCPT NPs treatment group, while the fluorescence signals in free HCPT treatment group were rather weakly (Figure 5). 3.7 In Vivo Blood Circulation and Biodistribution Before we applied nanoparticles in biomedicine, we need to clearly understand their in vivo blood circulation profile. To this end, mice bearing LLC tumor were i.v. injected with 1) free PS, 2) free HCPT, 3) CTPP NPs, and 4) CTPP/HCPT NPs. Periodically, blood samples were harvested and drug content in blood was determined. As shown in Figure 6, free drugs showed rapid clearance with half-lives were 0.82 h (PS) and 0.46 h (HCPT), respectively. On the contrary, CTPP/HCPT NPs displayed prolonged half-life of PS from 0.82 h to 5.39 h (6.57-fold), and HCPT from 0.46 h to 2.33 h (5.07-fold), respectively. The drugs biodistribution and organ clearance was also important for improving anticancer efficacies and reducing toxic. As depicted in Figure 7a and b, HCPT and PS levels in heart, lung


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and kidney was rather low (< 4 µg/g) at all time points, while HCPT and PS levels in spleen, liver and tumor tissues has an obvious early time point accumulation. Very interestingly, the HCPT and PS were rapidly cleared from liver and spleen at 24 h (< 6 µg/g). On the contrary, the clearance of HCPT and PS from tumor tissues was very slow owing to the EPR effect. This behavior is favorable for enhancing anticancer efficacy and reducing systemic toxicity. 3.8 In Vivo Anticancer Efficacy The outstanding performance in vitro promoted us to further explore anticancer efficiency in vivo. Comparative evaluation of different drug formulations on the antitumor efficacy was conducted on murine model (Figure 8a). The results showed that weakly inhibited tumor growth was found in free drugs group compared with PBS group. CTPP NPs exhibited slightly enhanced tumor suppressive effects. As for CTPP/HCPT NPs, the best tumor inhibition effect was achieved during the whole therapeutic period. Correspondingly, survival time of mice in CTPP/HCPT NPs group was remarkably prolonged (Figure 8b). The detailed data of in vivo anticancer efficacy indexes (TV, RTV, TGI and cures) were calculated and listed in Table 3. In addition, little change was found in body weight, revealing the low system toxicity (Figure 8c). 3.9 Evaluation of Adverse Effects Except for potent therapy efficiency, well-restrained adverse effect was also critical for successful chemotherapy. Generally, type-1 hypersensitivity reaction is the most common for many natural anticancer drugs. The IgE level was selected to evaluate type-1 hypersensitivity reactions owe to its important role in type-1 hypersensitivity reactions. After treatment with free drugs, the IgE level was significantly increased due to the poor water-solubility (Figure 8d). Excitingly, the IgE level of mice in nanoparticle groups was found no obvious change, suggesting the good biocompatibility. Meanwhile, the WBC and PLT counts were collected to


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evaluate the hematologic toxicity (Figure 8e and f). The results showed that the treatment with nanoparticle rather than free drugs could maintain the WBC numbers and PLT numbers in a normal range, suggesting that our nanoparticles could effectively avoid hematologic toxicity. 4. Conclusion In summary, a novel pH and redox dual-responsive polymeric nanoparticle CTPP/HCPT have successfully prepared for efficient drug delivery. These NPs showed typical spherical shape with relatively uniform size distribution, exhibited good biocompatibility, low hemolysis, long circulation time, and high cellular uptake. All these excellent properties revealed their great potential for efficient drug delivery. Importantly, these NPs displayed significant pH and redox dual responsiveness, leading to controlled drug payload release. In vitro experiment using CTPP/HCPT NPs showed excellent performance in inducing cancer cell death. Further investigation in vivo revealed that such dual-responsive CTPP/HCPT NPs could effectively inhibit tumor growth, without eliciting severe unfavorable adverse effects. Hence, we believed that the CTPP/HCPT NPs prepared here would have important role in cancer treatment.

ACKNOWLEDGMENT This study was supported by National Key R&D Program of China (2017YFF0207804), the Chinese Central Level Public Welfare Scientific Research Institutes Foundation for Basic Research & Development (562016Y-4687) and the National Natural Science Foundation of China (No. 21576029; 21406013). Supporting Information Available


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Table 1 Characterization of nanoparticles Samples

Size (nm)

ζ-potential (mV)

PDI

DLC of (wt%)

PS DLC of HCPT EE (wt%) (%)

CTPP NPs

116

-21.2 ± 4.82

0.18 24.7 ± 1.86

-

-

CTPP/HCPT NPs

144

- 23.7 ± 3.54

0.11 20.6 ± 1.67

22.8 ± 2.44

89.2

Table 2 In vitro cytotoxicity analysis (IC50, µg/mL) Samples

LLC cell

A549 cell

Free PS

11.24 ± 1.46

15.47 ± 1.17

Free HCPT

0.51 ± 0.08

3.36 ± 0.54

CTPP NPs

5.44 ± 0.27

10.78 ± 1.23

CTPP/HCPT NPs

0.28 ± 0.05

1.85 ± 0.09

Table 3 In vivo anticancer efficiency Samples

Mean TV (mm3)a

RTV a

TGI (%) a

Cures (%)b

PBS

4485 ± 1405

34.5 ± 11.9

0

0

Free PS

3250 ± 1005

25.4 ± 6.9

26.4

16.7

Free HCPT

2864 ± 873

22.1 ± 6.7

35.9

33.3

CTPP NPs

1612 ± 665

12.4 ± 5.2

64.1

50

CTPP/HCPT NPs

658 ± 184

5.1 ± 1.4

85.2

83.3

a

Mean tumor volume (TV), RTV, and %TGI data were taken at day 20. (By day 20, a

significant percentage of control animals were euthanized due to excess tumor burden.)

b

%

Cures were taken at day 22.


22

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Figure 1. Illustration of pH and redox dual-responsive CTPP/HCPT NPs for dually activated intracellular release of anticancer drugs.


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Figure 2. (a) TEM images and (b) DLS of CTPP/HCPT NPs; (c) in vitro stability of nanoparticles after 96 h storage; (d) in vitro PS release at different pH values; (e) in vitro HCPT release at different pH values with or without 10 mM GSH; (f) hemolysis experiments of different nanoparticles.


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Figure 3. In vitro cytotoxicity of LLC and A549 cells. Dose-dependent cell viability of (a) LLC and (b) A549 cells treated with PS, HCPT, CTPP NPs and CTPP/HCPT NPs (equivalent to PS or HCPT) for 72 h (n = 3, error bars represent standard deviation); time-dependent cell viability of (c) LLC and (d) A549 cells treated with different drug formulations for 24, 48, and 72 h (n = 3, error bars represent standard deviation).


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Figure 4. Cellular uptake of free HCPT and CTPP/HCPT NPs. (a) Histogram analysis and (b) mean fluorescence intensity were analyzed by flow cytometry.


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Figure 5. Confocal images of LLC cells incubated with free HCPT and CTPP/HCPT NPs at an equivalent HCPT concentration.


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Figure 6. (a) Blood circulation curves (b) half-lives of CPTT NPs and CTPP/HCPT NPs compared with free PS; (c) blood circulation curves (d) half-lives of CTPP/HCPT NPs compared with free HCPT.


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Figure 7. Biodistribution of HCPT (a) and PS (b) delivered by CTPP/HCPT NPs. CTPP/HCPT NPs were i.v. administered at 20 mg HCPT or PS/kg into LLC tumor bearing C57BL/6 mice, and total HCPT and PS in the tissues were extracted at different times (4 h, 12 h, 24 h and 72 h) and measured by HPLC.


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Figure 8. In vivo antitumor activity of free PS, free HCPT, and nanoparticles in the subcutaneous mouse model of LLC. (a) Tumor volumes of mice during treatment with different groups; (b) survival of mice in different treatments; (c) body weight of mice in different groups; (d) IgE levels, (e) WBC changes, and (f) PLT changes in bloods after treated with different drug formulations.


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For Table of Contents Use Only Self-assembled pH and redox dual responsive carboxymethylcellulose-based polymeric nanoparticles for efficient anticancer drugs co-delivery

Ke-Feng Liu,a Yan-Xue Liu,a Chun-Xiao Li,a Lu-Ying Wang,a Jing Liu,a Jian-Du Lei*a,b

a

b

Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing 100083, P. R. China

Chemical and Biomolecular Engineering Department, University of California-Los Angeles, USA, CA 90095


31


Illustration of pH and redox dual-responsive CTPP/HCPT NPs for dually activated intracellular release of anticancer drugs 66x54mm (300 x 300 DPI)


Page 32 of 32

Self-Assembled pH and Redox Dual Responsive

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