Smart Nanoparticles Undergo Phase Transition for Enhanced Cellular

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Smart Nanoparticles Undergo Phase-transition for Enhanced Cellular Uptake and Subsequent Intracellular Drug Release in Tumor Microenvironment Guihua Ye, Yajun Jiang, Xiaoying Yang, Hongxiang Hu, Beibei Wang, Lu Sun, Victor C. Yang, Duxin Sun, and Wei Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b15978 • Publication Date (Web): 20 Dec 2017 Downloaded from http://pubs.acs.org on December 20, 2017

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ACS Applied Materials & Interfaces

Smart Nanoparticles Undergo Phase-transition for Enhanced Cellular Uptake and Subsequent Intracellular Drug Release in Tumor Microenvironment

Guihua Ye1, Yajun Jiang1, Xiaoying Yang1, Hongxiang Hu2, Beibei Wang1, Lu Sun1, Victor C. Yang1,2, Duxin Sun2, Wei Gao1,2,*

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Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, PR China. 2 University of Michigan, College of Pharmacy, 428 Church St, Ann Arbor, MI 48108, USA. *Corresponding author: Wei Gao E-mail: [email protected]; Tel/Fax: 17347735423

Abstract：：The inefficient cellular uptake and intracellular drug release at tumor site are two major obstacles limiting the antitumor efficacy of nanoparticle delivery systems. To overcome both problems, we designed a smart nanoparticle undergoes phase transition in tumor microenvironment. The smart nanoparticle is generated using a lipid–polypetide hybrid nanoparticle, which comprises a PEGylated lipid monolayer shell and a pH sensitive hydrophobic poly-L-histidine (PHIS) core, and is loaded with antitumor drug doxorubicin (DOX). The smart nanoparticle undergoes a two-step phase-transition at two different pHs in tumor microenvironment: (i) At tumor microenvironment (pHe: 7.0 ~ 6.5), the smart nanoparticle swells, its surface potential turns from negative to neutral, facilitating the cellular uptake; (ii) After internalization, at acid endolysosome (pHendo: 6.5 ~ 4.5), the smart nanoparticle dissociates and induces endolysosome escape to release DOX into cytoplasm. In addition, a tumor penetrating peptide iNRG was modified on the surface of the smart nanoparticles as a tumor target moiety. The in vitro studies demonstrated that the iNGR modified smart nanoparticles promoted cellular uptake at acidic environment (pH 6.8). The in vivo studies showed that iNGR modified smart nanoparticles exerted more potent antitumor efficacy against the late stage aggressive breast carcinoma than free DOX. These data suggest that the smart nanoparticles may serve as a promising delivery system for sequential uptake and intracellular drug release of antitumor agents. The easy preparation of these smart nanoparticles may also provide advantages in future manufacture for clinical trials and clinical use. Keywords：： Sequential drug delivery systems; pH sensitive; tumor target; poly-L-histidine；iNGR

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1. Introduction Delivering chemotherapeutic agents by nanoparticles is considered to be a promising strategy for enhancing their therapeutic response and reducing side effects. The first FDA-approved liposomal drug, PEGylated liposomal doxorubicin, known as Doxil®, was released on the market in 1995, and is being extensively used in clinic over 20 years 1. However, clinical data from different trials during these years reveal conflict results on their therapeutic benefits over free doxorubicin (DOX); several clinical studies demonstrate that PEGylated liposomal DOX does not exhibit advanced antitumor efficacy than free DOX in long-term treatments 2-7. Inefficient cellular uptake and intracellular drug release at tumor site are two major impediments limiting the antitumor efficacy of liposomal DOX as well as other kinds of nanoparticles 8. Especially, when PEG was introduced to coat nanoparticles, a commonly used method to realize long circulation, the highly hydrophilic PEG corona with large steric badly hinders the nanoparticles interaction with negatively charged cell membrane and suppresses their cellular uptake 9-10. Moreover, despite some of the particles are successfully endocytosed by tumor cells, most of them are trapped in acid endolysosomes and unable to release drugs into cytoplasm 11. One of the most promising strategies to promote tumor specific cellular uptake and intracellular drug delivery is to design ‘smart’ bioresponsive nanoparticles, which undergo particle transition or drug release in response to tumor microenvironment (TME) signals (redox potential, acid pH, or unique enzymatic activity)12-13. Acid or enzyme triggered deshielding of the PEG chain or layer at TME has been proved a better cellular uptake10. Charge conversional nanoparticles carry negative charge for enhanced circulation, then transform to positive at TME, are also considered to be an effective method to increase cellular uptake, since surface charge crucially affects tumor specific cellular uptake and intracellular delivery10. For instance, the acidification of cancerous sites (pH of tumor microenvironment, pHe: 7.0 ~ 6.5) and the intracellular endolysosomes (pH of endolysosome, pHendo: 6.5~4.5) create an ideal pH gradient for controlling the delivery of drugs. Du et.al have prepared a surface-charge switchable nanoparticle, which is negatively charged at physiological pH (~7.4), but undergoes charge reversion from negative to positive at pHe to enhance cellular internalization14. To facilitate intracellular drug delivery, Bea et al. have developed a micellar system using polyhistine (PHIS), a special pH sensitive materials that can trigger endolysosome escape via the so-called “proton sponge” effect. The micellar system was fabricated by PHIS-PEG2000 and poly(l-lactide)-PEG2000 (PLLA-PEG2000), and the triggering pH is tailored to 6.0 by optimizing the ratio of the two polymers. The micelle has an ability of endolysosome escape, and shows a great enhancement of intracellular drug release15. In order to solve the cellular uptake and intracellular delivery problems at the same time, several sequential or step-by-step drug delivery systems have recently been developed16. For instance, a multistage pH-responsive liposomes containing a synthesized smart lipid (1,5-dioctadecyl-L-glutamyl 2-histidyl-hexahydrobenzoic acid, HHG2C18) are prepared, which is capable of switching the surface charge from highly negative to positive at tumor microenvironment to enhance cellular uptake, and sequentially causing endolysosomal bursting after endocytosis17. The sequential drug delivery systems exhibit a promising efficacy in animal experiments; however, they often contain complex structures or require multiple steps or chemical synthesis in preparations, which are not appropriate for large-scale manufacture. In this study, we aim to solve both the cellular uptake and intracellular drug release problems using a simply-structured pH sensitive nanoparticle. Poly-L-histidine (PHIS) is employed in the 2


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nanoparticle as a pH sensitive switch. PHIS is a widely used biomaterial with good biocompatibility and biodegradability 18. The imidazole groups in its side chain undergo protonation at weak acidic pH, allowing PHIS chain transits from hydrophobic neutral to hydrophilic cationic form. When PHIS delivers drugs or biomacromolecules into acid endolysosome, it can trigger endolysosome escape and release the cargoes into cytoplasm19. Recently, Iwasaki. et al has demonstrated that PHIS is also a highly efficient cell-penetrating peptide that can be applied as a vector to facilitate cellular uptake of the cargoes20. The modification of tumor specific antibody or ligand on the surface of nanoparticles, known as active targeting strategy, improves the tumor accumulation of nanoparticles 21. Tumor penetrating peptide iNRG (CRNGRGPDC) is a recent developed target moiety. iNRG contains two sequence motifs: a tumor-homing motif NRG that binds to CD13 on tumor vessels and tumor cells; and a cryptic CendR motif that induces greater tumor penetration of coupled nanoparticles 22. Previous researches from several individual groups have demonstrated that iNRG enhances tumor accumulation and penetration of its anchored nanoparticle 23-25. Herein, we designed a smart lipid–polypeptide hybrid nanoparticle (LPN) with a simple structure, and successfully prepared via a single-step nanoprecipitation method, potentially suitable for scale-up 26. As illustrated in Fig 1, the LPN consists of a PEGylated lipid monolayer shell (for long circulation and good biocompability), a pH sensitive poly-L-histidine core (for pH responsive), and loaded with antitumor drug DOX. A tumor specific ligand iNGR was modified on the surface of the particles for tumor targeting. Particularly, we observed that the LPN undergoes a two-step phase-transition at tumor acid pH: (i) At tumor microenvironment (pHe: 7.0 ~ 6.5), the protonated segments of the polyhistine chain moved to the surface of the particle, the particle swells, and its surface potential turns from negative to neutral or positive, facilitating the cellular uptake; simultaneously, a portion of encapsulated DOX releases from the particle further increasing the cellular uptake (ii) After internalization, at acid endolysosome (pHendo: 6.5 ~ 4.5), the particle dissociates and induces endolysosome escape to release drugs into cytoplasm. To evaluate the therapeutic efficacy of iNRG modified LPN (iNRG-LPN), we established 4T-1 murine breast tumor model that represents the late stage aggressive breast carcinoma in clinic, poorly responding to traditional chemotherapy such as free DOX treatment. By using the tumor model, we compared the cellular uptake, cytotoxicity, in vivo antitumor efficacy and systemic toxicity of DOX loaded iNRG-LPN with free DOX to evaluate the improvements of therapeutic response.

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Figure 1 Schematic illustration of pH triggered two step phase-transition of iNGR-LPNs (A) and the delivery of antitumor drug DOX (B). The DOX loaded iNGR-LPNs was first accumulated in tumor site driven by EPR effect and iNGR specific binding to CD13 on tumor vessels. At tumor microenvironment (pHe: 7.0~6.5), the particle undergoes first phase transition to facilitate nanoparticle uptake, and simultaneously releasing a portion of encapsulated DOX; After internalization, at acid endolysosome (pHendo: 6.5~4.5), the particle dissociates and induces endolysosome escape to release DOX into cytoplasm, then free DOX enters into nucleus exerting cytotoxicity. 2. Materials and Methods 2.1 Materials Polyhistidine (PHIS, Mw: 3035, >95% by HPLC) was synthesized by GL Biochem Co., Ltd (Shanghai, China). CRNGRGPDC (iNGR, 97.62% by HPLC) was synthesized by ChinaPeptides Co., Ltd. (Shanghai, China). DSPE-PEG (PEG, Mw:2000), DSPE-PEG-NHS (PEG, Mw:2000), Egg yolk phosphatidylcholine (EPC) and Cholesterol (CHO) were purchased from the Advanced Vehicle Technology Pharmaceutical L.T.D. Co. (Shanghai, China). Doxorubicin hydrochloride (DOX·HCl) was purchased from Melone Pharmaceutical Co., Ltd. (Dalian, China). 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) was purchased from Molecular Probes Inc. (Eugene, OR, USA). Triethylamine (TEA) was purchased from Shanghai Titanchem.Co.Ltd. (Shanghai, China). Na2B4O7·10H2O and anhydrous dimethylsulfoxide (DMSO) were purchased from Aladdin Industrial Corporation (Qingdao, China). All other agents used in this study are of analytical grade. The mouse mammary tumor cell line (4T1) was obtained from the Institute of Basic Medical Science at the Chinese Academy of Medical Science (Beijing, China). Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) 4


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and 1% penicillin-streptomycin at 37 °C with 5% CO2 in humidified environment. DMEM and FBS were provided by Biological Industries USA, Inc. (Connecticut, USA). Trypsin-EDTA solution and penicillin-streptomycin were purchased from Basal Media Ltd. (Shanghai, China). And 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Healthy female BALB/c nude mice (18-20 g, approximately 6 weeks old) were supplied by Peking University Health Science Center. Before the test, mice were feed at 25 °C and 55% of humidity for 1 week, with free access to standard water and food. All animal experiments conducted in this study were performed under the National Institutes of Health Guide for the Care and Use of Laboratory Animals. 2.2 Methods 2.2.1 Preparation of the lipid–polymer hybrid nanoparticles (LPNs) The LPNs was synthesized by a single-step nanoprecipitation method26. In brief, PHIS (4.1 mg) was dissolved in 1 mL DMSO as organic phase. EPC and DSPE-PEG (7:3, molar ratio, totally 5.4 mg) were dissolved in Na2B4O7 (2 mM) solution (pH 9.0) containing 4% ethanol and heated to 65 °C. The PHIS DMSO solution was then added into the preheated EPC/DSPE-PEG solution dropwise (1 mL/min) under violently stirring to obtain a mixture (DMSO: Na2B4O7 solution: 1:10, v/v), following continuous stirring for 2 h at room temperature to allow spontaneously self-assembly. Then the solution was dialyzed against Na2B4O7 solution (2 mM) with cellulose ester membranes (molecular weight cut off 3500 Da, Spectrum Medical Industries, Rancho Dominguez, CA) for 24 h. The final LPNs solution was obtained and stored at 4 °C. The iNGR modified LPNs was prepared with the same procedures, expect replacing a portion of DSPE-PEG with iNGR-DSPE-PEG (DSPE-PEG: iNGR-DSPE-PEG = 7:3, molar ratio). To prepare the doxorubicin (DOX) loaded LPNs, DOX·HCl (1 mg) was first reacted with TEA in DMSO for 2 h to eliminate HCl, and then added dropwise to the PHIS solution under continuously stirring for another 2 h. The rest preparation process was the same as described above. For in vivo imaging test, the near-infrared fluorescent probe DiR was loaded into LPNs or iNGR-LPNs. The preparation process was the same as that of LPNs except for the addition of DiR in the PHIS solution. 2.2.2 Synthesis of iNGR-PEG-DSPE To synthesis iNGR-PEG-DSPE, iNGR was conjugated to the terminal NHS group of DSPE-PEG2000-NHS according to the previous reports27. Briefly, iNGR and DSPE-PEG2000-NHS (1:1.5, molar ratio) were dissolved in anhydrous DMSO at pH 8.0-8.5 adjusted by trimethylamine (TEA). The reaction solution was continuously stirred under nitrogen gas for 5 days at 30 °C. After the reaction, the solution was dialyzed against deionized water for 48 h with cellulose ester membranes (molecular weight cut off 3500 Da, Spectrum Medical Industries, Rancho Dominguez, CA) to remove the unreacted iNGR and DSPE-PEG-NHS. The solution was then lyophilized and stored at -20 °C. The final product was confirmed by an Axima-CFR plus (Kratos Analytical Ltd, Manchester, UK) mass spectrometer. 2.2.3 Characterization of DOX loaded LPNs and iNGR-LPNs Particle size distribution and zeta potential The particle size distribution and surface charge of LPNs and iNGR-LPNs were determined by dynamic light scattering (DLS) analysis using a Malvern Zetasizer Nano ZS (Malvern, UK) at 25 °C. 5



The morphology of LPNs and iNGR-LPNs was observed by a transmission electron microscope (TEM) (Hitachi HT7700, Japan). The TEM samples were prepared by adding the iNGR-LPNs solution onto a carbon-coated copper grid and stained for 10 min with phosphotungstic acid solution (2%) at room temperature and dried in the air. Encapsulation and loading efficiencies (EE% and LE%) The dialysis method was used in the preparation of the LPNs to remove free DOX as described in 2.2.1. The amount of DOX after dialysis was measured to calculate EE% and LE%. The concentration of DOX was quantified by a UV-VIS spectrophotometer (HITACHI, Japan) at 488 nm. The EE% and LE% of the LPNs and iNGR-LPNs were calculated according to the following formulas: EE% = (The amount of DOX in LPNs after dialysis/the amount of DOX input) × 100% LE% = (The amount of DOX in LPNs after dialysis/the weight of DOX loaded LPNs) × 100% 2.2.5 The observation of pH induced phase transition of the LPNs The variation of pyrene fluorescence that loaded into the LPNs can reflect the dissociation process of the LPNs28. Test tubes were first filled with pyrene acetone solution and dried under N2 avoiding light. LPNs solution was added to the tubes and sonicated 30 min and placed at room temperature for 1 h. The HCl solution was added dropwise to adjust the pH from 7.5 to 4.0. At different pH, the fluorescence emission spectrum of the pyrene was measured with excitation wavelength set at 339 nm. The intensity ratio of the first and the third peaks (I373/I383) in the emission spectrum was recorded. The phase transition process of the LPNs with pH can also be tracked by measuring the variation of zeta potential, size distribution and morphology. The HCl solution was added dropwise to the LPNs solution to adjust the pH. At each preset pH, the zeta potential and size of the particles were measured by a dynamic light scattering (DLS) analysis using a Malvern Zetasizer Nano ZS (Malvern, UK) at 25 °C, and morphology of the particles were observed by a transmission electron microscope (TEM) (Hitachi HT7700, Japan). 2.2.6 The in vitro drug release test at different pH The DOX release profile from the LPNs was tested by a dialysis method. Briefly, LPNs and iNGR-LPNs were added into a dialysis bag (3500 Da, Shanghai Jinsui Bio-Technology Co., Ltd) and placed into 30 mL PBS solution (10 mM, pH 7.4, 6.5 or 5.0) as dialysis medium under continuous shaking at 37 °C for 24 h. At each time point (0.5, 1, 2, 4, 8, 12 and 24 h), 1 mL of the outer phase was withdrawn and replaced with equal volume of dialysis medium. The concentration of DOX was determined by a high-performance liquid chromatography (HPLC) system (Agilent 1260 Infinity, USA) with an ODS column (Agilent ZORBAX SB-C18, 4.6×250mm, 5µm) and an UV detector at 488 nm. The flow rate was 1 mL/min. The mobile phase consisted of water (1.44 g SDS, 0.68 mL H3PO4 in 500 mL deionized water), methanol and acetonitrile (40:5:50, v/v). 2.2.7 Flow cytometry analysis The cellular internalization of various DOX formulations was analyzed by flow cytometry. 4T1 cells (approximately 1×105) were seeded into 12-well plates and incubated for 24 h. Then the medium was replaced with culture medium (pH 6.8) containing free DOX, DOX loaded LPNs or iNGR-LPNs at the concentration of 5 µg/mL DOX. After 2 h incubation, the cells were harvested by trypsin-EDTA, washed three times with cold PBS solution and examined by a FACScan flow cytometer (BD FACSVerse, USA) with excitation and detection wavelengths set at 488 nm and 6


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575 nm. The number of collected cells was over 10 000 for each sample. 2.2.8 In vitro cytotoxicity assays The in vitro cytotoxicity of various DOX formulations against 4T1 cells were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Briefly, a volume of 100 µL cell suspension (approximately 8 000 cells) was added into 96-well plates and incubated for 24 h. The cells were treated with different concentrations of free DOX, DOX loaded LPNs or iNGR-LPNs (DOX concentrations from 0.01 to 10 µg/mL diluted by DMEM at pH 6.5). After 24 h incubation, the cells were washed three times with PBS. A volume of 100 µL DMEM containing 20 µL MTT (5 mg/mL in PBS) was added into each well and incubated for another 4 h avoiding light. Finally, the medium was replaced by 200 µL DMSO and the plate was shaken via an IS-RDD3 Thermostat oscillator (Texas, USA) at 100 rpm for 10 min. The absorbance of each well was measured at 570 nm by a Thermo MULTISCAN GO microplate reader (Thermo, UK). The cell inhibition rate was calculated as the percentage of cell viability comparing to the control group; and the concentration of DOX causing 50% cell inhibition (IC50) was calculated for each group by linear regression analysis. 2.2.9 In vivo imaging test The biodistribution of LPNs and iNGR-LPNs were determined using an in vivo image method. First, 4T1 cells were subcutaneous inoculated into the right flanks of female BALB/c nude mice. When the tumor volume reached circa 100 mm3, each mouse was injected via tail vein with near infrared fluorescence probe DiR loaded LPNs or iNGR-LPNs (0.4 µg/ 100 µL). At each time point (1, 3, 6, 8, 20, 24, 36 and 48 h), the mice were anesthetized with isoflurane, and the fluorescent and X-ray images were captured by an in vivo imaging system (FX Pro; Kodak, Rochester, NY, USA). After 48 h, mice were euthanized. Tumors and major organs were excised and imaged. 2.2.10 In vivo antitumor efficacy and systemic toxicity tests The in vivo test was conducted on BALB/c mice bearing 4T1 murine mammary. First, 4T1 cells with the number of approximately 1×106 for each mouse were subcutaneous inoculated in the right flanks of female BALB/c mice. When tumors reached about circa 100 mm3, the mice were randomly divided into six groups (n =8) and administrated via tail vein with one of the following regimens at day 1, 4, 7, 11, 14: Group 1 =PBS (pH 7.4); Group 2 = free DOX (5 mg/kg in DOX); Group 4 = LPNs (10 mg/kg in DOX); Group 5 = iNGR-LPNs (10 mg/kg in DOX). Group 1 was served as a control group. The survival rate of the animals (n = 6, minus the two mice scarified for the histological analysis) was calculated for each group until all the animal dead. The survival curve was drawn, and the median survival time of each group was calculated using the Log rank test with GraphPad V5.0. The mice weight was measured at every other day after the treatments. For histological analysis, we randomly chose two mice from each group and sacrificed them on day 25 to collect the tumor and heart for histological section. The heart sections were stained with hematoxylin and eosin (H&E) to evaluate the DOX related cardiotoxicity. For the tumor sections, CD31 stain was preformed to measure the tumor microvessel density, and Ki 67 stain was used to evaluate tumor cell proliferation. All histopathologic specimens were observed via a light microscopy (Wuhan Goodbio technology CO., LTD, Wuhan, China). 2.2.11 Statistical analysis All the tests were performed at least three times unless otherwise specified. Data was reported as means ± standard deviation (SD) unless otherwise specified. The differences among groups were evaluated by a two-tailed Student's t-test analysis. A p value less than 0.05 or 0.01 was considered 7



to be statistically significant or highly significant. 3. Results and discussion 3.1 Preparation and characterization of DOX loaded iNGR-LPNs The LPNs was successfully prepared via a single-step nanoprecipitation method, also being called as a modified nanoprecipitation method, which is previously used to prepared the similar-structured polymer core–lipid shell nanoparticles26, 29. This method avoids time-consuming preparation steps, providing advantages for large-scaled manufacture26. Briefly, lipids (EPC and DSPE-PEG) were dispersed in Na2B4O7 solution at 65 °C to ensure proper dispersion. The PHIS and DOX solution was then added into the preheated EPC/DSPE-PEG solution dropwise under violently stirring. As the solubility of PHIS and DOX are low in Na2B4O7 solution, they precipitate together to form the hydrophobic core. Simultaneously, lipids (EPC and DSPE-PEG) pre-dispersed in Na2B4O7 solution self-assemble around the hydrophobic core to form a lipid monolayer covered by a PEG shell, the process is driving by the hydrophobic interaction between hydrophobic chain of the lipids and the core (Fig 2A)29-30. The mixture was then dialyzed in Na2B4O7 buffer (pH 9.0) buffer to remove DSMO and free DOX that were not encapsulated into the particles. The Na2B4O7 buffer was used as dialysis medium according to the previous reports on preparation polyhistine based nanoparticles30-31. We compared the Na2B4O7 buffer with PBS buffer, the drug loading efficiency and stability was higher by using Na2B4O7 buffer than PBS buffer. When used in body, we either diluted the nanoparticles solution or changed the medium to PBS or saline by ultrafiltration. The formulation of DOX loaded LPNs was optimized as DSPE-PEG 3.2 mg/mL, EPC 2.2 mg/mL, PHIS 4.1 mg/mL and DOX 1 mg/mL. The obtained LPNs are around 100 nm with a narrow size distribution, and negatively charged at pH 7.4 (Fig 2B and Tab 1). The representative TEM photograph shows a typical spherical shape (Fig 2C). DOX was encapsulated into LPNs with high encapsulation efficiency (EE%: 84.59% ± 2.19%) and loading efficiency (LE%: 9.08% ± 0.33%) (Tab 1).

Figure 2 The Preparation and characterization of DOX loaded LPNs. (A) The schematic illustration. The particle size distributions (B) and the typical TEM image (C) of the optimized 8


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DOX loaded LPNs formulation. (D1) The influence of PEGylation and lipid-to-PHIS ratio on the size (D2) and pH sensitivity (D3) of the LPNs. F1, F2, F3, F4, F5 and F6 represents six formulations of LPNs.For each formulation, the ratio of DOX to the total weight of materials is 1:9.5. Table 1 Characterization of DOX loaded LPNs and iNGR-LPNs (n=3) Empty LPNs

LPNs

iNGR-LPNs

Size(nm, Z-average) PDI

114.59 ± 6.06 0.26 ± 0.07

124.14 ± 8.44 0.28 ± 0.05

116.73 ± 4.18 0.25 ± 0.02

Zeta potential (mV) EE (%) LE (%)

-7.99±0.43

-7.96 ± 1.34 84.59 ±2.19 9.08 ± 0.33

-8.12 ± 1.58 87.98 ± 3.62 7.64 ± 0.70

The PEGylation and lipid-to-PHIS mass ratio are two important factors to form an ideal LPNs. As shown in Fig 2D2, the formulations (F1, F2, F3) without the addition of DSPE-PEG shows an obvious aggregation, as indicated by the large average diameter (620 ~740 nm) and a wide size distribution (PDI: 0.56 ~ 0.95). An increase in total lipid (EPC and DSPE-PEG) to PHIS mass ratio from 25% to 75% does not prevent the aggregation, which demonstrates that even thick lipid layer to cover the hydrophobic core is insufficient to stabilize LPNs. On the contrary, when DSPE-PEG (30% of total lipid) was added into the formulations, the size of LPNs (F4, F5, F6) turned into 100~200 nm with a narrow size distribution. It indicates that surface PEGylation plays a crucial role in stabilizing LPNs from aggregation thus resulting in smaller and uniform nanoparticles. Next we tested the pH sensitivity of F4, F5 and F6 by measuring the change of zeta potential. As shown in Fig 2D3, all the three formulations show negative charged at pH 7.4, suggesting a good coverage of PHIS core by PEGylated lipid membrane. However, only F6 (total lipid: PHIS=1:3, mass ratio) shows an obvious charge reversal at pH ~6.5; whereas F4 and F5 with thicker lipid layer (total lipid: PHIS mass ratio= 1:1 or 3:1, mass ratio) merely undergo slight potential increase with pH and remain negative at pH 4.5. The results suggest the pH sensitive property of LPNs requires a proper thickness of lipid layer to make a good cover of hydrophobic core at physiological pH, while at the same time allowing the remolding by protonated PHIS at acidic pH.

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Figure 3 The pH stimulated two-step phase-transition of LPNs. (A) The change of pyrene fluorescence intensity (I373/I383) with pH. Pyrene was preloaded into LPNs. (B) Zeta potential of LPNs at different time point at pH 5.5, 6.5 and 7.4. (C) The size distributions and TEM images (D) of LPNs at pH 5.5, 6.5 and 7.4. The scale bar is 200 nm. 3.2 The pH stimulated phase-transition of LPNs Most of the reported PHIS formed pH sensitive micelles or nanoparticles undergo pH stimulated transition at pH 6.5~4.5 (the pH of endolysosome, pHendo) and exert the proton sponge effect to facilitate the endolysosome escape 15, 32. Herein, the LPNs hold a special two-step phase transition property at acidic pH, thus not only it can induce endolysosome escape after endocytosis, but also facilitate the cellular uptake of nanoparticles at tumor microenvironment, as illustrated in Fig 1A. To prove the hypothesis, we first monitored the pyrene fluorescence intensity variation when pH drops. The pyrene probe was encapsulated into the LPNs. The pyrene can sense the micropolarity change inside the LPNs and reflect the dissociation of the particles via the intensity ratio of the first and the third peaks (I373/I383) in the emission spectra profile 28. As shown in Fig 3A, the I373/I383 value is consistent at pH 7.4~6.5 but undergoes a sharp decline at pH 6.5~4.5. The result demonstrated that the LPNs remained integrated at pH 7.4~6.5, but gradually dissociated at a pH lower than 6.5. To further prove the nanoparticle’s phase change, we tested the size distribution and morphology of LPNs at pH 7.4, 6.5 or 5.5, respectively. As shown in Fig 3C and D, at pH 7.4, 10


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LPNs remained stable as uniform spherical particles with approximately 100 nm in diameters. When the medium pH dropped to 6.5, the pH of tumor environment (pHe), the average diameters of the particles increased to 141±32 nm. From the TEM image, we can see that the particles remain in spherical shape but with relatively larger size and less uniform distribution at pH 6.5 than that at pH 7.4. When further dropped the medium pH to 5.5 (pHendo), nonuniform size distribution was observed; large particles as well as irregular aggregations are seen in TEM images (Fig 3C). The results indicate that LPNs dissociate after swelling at pH 5.5. Interestingly, as shown in Fig 2D3 (line F3), while slowly adding hydrogen ions into the medium, the potential of the LPNs showed a sharp increase at pH 7.4~6.5, and converted to positive at around pH 6.5 (pHe), then continuously increased until pH 4.5. We conducted the same experiment on a DOX-liposome, which had no pH stimulated property. As expected, the zeta potential of the DOX-liposome remained consistent when pH dropped (Fig S1). To determine whether the phase transition of the LPNs is a quick or graduate process, we tested the particle potential changes with time. The zero point was recorded as soon as the LPNs were added to the buffer at pH 7.4, 6.5 or 5.5. As shown in Fig 3B, the potential of changes at the first time point at pH 6.5 or 5.5, indicating a very fast transition process. At pH 6.5, the potential remains stable after the transition, that further proves that the particle can remain in this stage until pH further drops. To summarize, the results suggest that the LPNs undergo a two-step phase transition at acidic pH, as illustrated in Fig 1A. First, at pH 7.0~6.5 (pHe), PHIS in the core of the LPNs partially protonates, and turns from hydrophobic to amphiphilic, which drives the particle swelling. Simultaneously, the hydrophilic and cationic part of PHIS chain moves to the surface of the particle resulting an increase in zeta potential. The LPNs do not dissociate at this pH due to the protection of the lipid layer. Second, at pH 6.5~4.5, the PHIS completely protonates and interrupts the structure of lipid layer, making LPNs dissociated. We hypothesized that the special two-step pH responsive phase transition property of LPNs may facilitate cellular uptake and intracellular drug release at tumor site. We conducted the following cellular uptake and cytotoxicity test to prove the hypothesis.

Figure 4 The modification of LPNs with iNGR. (A) Synthesis scheme of DSPE-PEG-iNGR; (B) 11



MALDI-TOF mass spectrum of DSPE-PEG-iNGR. 3.3 The modification of LPNs with iNGR ligand To further improve the tumor accumulation, iNRG was modified on the surface of LPNs. First, iNRG was reacted with NHS-DSPE-PEG to form iNGR-DSPE-PEG conjugate (Fig 4A). The final product was identified by an Axima-CFR plus (Kratos Analytical Ltd, Manchester, UK) mass spectrometer. As shown in Fig 4B, the peak of 3961 indicates the molecular weight of iNGR-DSPE-PEG. The DOX loaded iNGR modified LPNs (iNGR-LPNs) were prepared by a similar protocol as DOX loaded LPNs, except the substitution a portion of DSPE-PEG to iNGR-DSPE-PEG. As shown in Tab 1, comparing to non-modified LPNs, the iNGR-LPNs exhibit similar size distribution, potential, as well as encapsulation and loading efficiency. 3.4 The pH stimulated drug release of LPNs The in vitro drug release test of DOX loaded LPNs, iNGR-LPNs was performed at pH 7.4, 6.5 and 5.5 for 24 h. As shown in Fig 5A, iNGR-LPNs show a pH stimulated drug release profile. At pH 7.4, DOX gradually and slowly release from iNGR-LPNs, and the accumulated released drug is only 19.32% during 24 h. When changing the medium pH to 6.5 (pHe), a quick release of drug is observed at the first four hours, and 53.83 % of drug is released during 24 h, which can be attributed to the swelling of the nanoparticle as described in part 3.2. The nature of the DOX molecules also contributes to the drug release here. Because DOX is loaded into the LPNs in the form of hydrophobic DOX base, and it protonates and turns into a more hydrophilic molecular at slight acidic pH, making it more easily to release from the swelling particles. When further decreasing medium pH to 5.5 (pHendo), iNGR-LPNs shows a burst drug release at first two hours (over 50 % of drugs released), and over 80% of drug released at 24 h because of the particle dissociation. Additionally, LPNs exhibit similar pH responsive release profile as, as shown in Fig 5B, indicating that modification of iNGR does not influence the pH sensitive releasing property of the nanoparticles.

Figure 5 The pH dependent drug release of LPNs. The profiles of DOX released from iNGR-LPNs (A) and LPNs (B) at pH 5.5, 6.5 and 7.4. 12


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3.5 Cellular uptake of various DOX formulations The cellular uptake of free DOX and DOX loaded LPNs, iNGR-LPNs was evaluated by flow cytometry analysis on breast tumor 4T-1 cells. We chose 4T-1 cell line because it represents the late stage aggressive breast carcinoma in clinic, which is difficult to treat in clinic by conventional chemotherapeutics. Moreover, it overexpresses aminopeptidase N (APN/CD13) receptor that can be recognized by iNGR. The pH of the culture medium was adjusted to 7.4 or 6.8 to simulate the physiological or tumor microenvironment. As shown in Fig 6A to C, by comparing the cellular uptake of LPNs at pH 7.4 and 6.8, we can conclude that the cellular uptake of LPNs at pH 6.8 is significantly higher than that at 7.4 (P

Smart Nanoparticles Undergo Phase Transition for Enhanced Cellular

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