Biodegradable Cyclomatrix Polyphosphazene Nanoparticles: A Novel

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Biological and Medical Applications of Materials and Interfaces

Biodegradable Cyclomatrix Polyphosphazene Nanoparticles: a Novel pH-Responsive Drug Self-Framed Delivery System Shenglei Hou, Shuangshuang Chen, Yuan Dong, Su Gao, Bangshang Zhu, and Qinghua Lu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06114 • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

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ACS Applied Materials & Interfaces

Biodegradable Cyclomatrix Polyphosphazene Nanoparticles: a Novel pH-Responsive Drug SelfFramed Delivery System Shenglei Hou, a Shuangshuang Chen, b Yuan Dong, a Su Gao, a Bangshang Zhu, a and Qinghua Lu. a

a

*

School of Chemical Science and Engineering, The State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, Shanghai, 200240. China. E-mail: [email protected]

b

School of Chemical Science and Engineering, Tongji University, Shanghai, 200092. China.

KEYWORDS: polyphosphazene, nanoparticles, biodegradable, self-framed delivery, tumor therapy

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ABSTRACT. Traditional drug delivery systems suffer from low drug-loading and relatively weak therapeutic efficacy, so development of new drug delivery systems with high-efficiency has become more urgent. In this report, a novel-innovative drug delivery strategy, namely drug self-framed delivery systems (DSFDS), is prepared via using anti-cancer drugs as polymer frame without using any carriers. The drug molecules (exemplified by doxorubicin) containing more than

two

nucleophilic

functional

groups

(diols/diamines)

directly

reacted

with

hexachlorocyclotriphosphazene via mild precipitation polycondensation under ambient conditions, forming biocompatible drug self-framed delivery nanoparticles. Due to the covalent bonding of the drug molecules, DSFD nanoparticles (DSFDs) with super high drug-loading were stable in the circulation during delivery. However, sustained release of drug in the acidic environment within cells endowed DSFDs with long-term anticancer therapeutic efficacy. This strategy is applicable for diverse hydrophilic and hydrophobic drugs and may be a new platform for designing high drug-loading and release-controllable drug delivery systems.

1. Introduction Development of effective cancer treatments is an urgent issue in modern medicine as a consequence of the sharply increasing number of deaths caused by a variety of malignancies. Among various cancer therapies, chemotherapy is vitally important contributing to the highefficiency in killing tumor cells.1 Unfortunately, the lethality of cancer is still high. Apart from the lack of specific drugs, low efficiency of drug delivery approaches is also a limitation

2, 3

In

the past, chemotherapy based on small molecule anticancer drugs has been restricted by many problems, including low water solubility,4 short circulation time and low accumulation in tumor tissue.5 To address these problems, the free small drug molecules were encapsulated in various nano-carrier systems,

6-9

which has been demonstrated to be useful because of the enhanced 2 ACS Paragon Plus Environment

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permeability and retention (EPR) effect.10 However, these nano-carriers themselves, organic carriers such as dendrimers,11, 12 polymer micelles or prodrug micelles,13-16 and liposomes,17-22 and inorganic carriers such as mesoporous silica nanoparticles (NPs),23-27 nanobubbles,28, 29 and carbon nanotubes,30-35 do not usually have therapeutic efficacy against cancer. The use of nanocarriers has suffered from several drawbacks, including higher metabolic load on the kidneys, relatively low drug carrying capacity and laborious preparation processes. For example, drugloading capacities were often below 10% (w/w), which was not satisfactory for most cancer therapies.36-38 Furthermore, the drawbacks of nano-carries limited their widespread application. Recently, drug self-delivery systems (DSDS), which are constructed from the active anticancer drugs themselves to form delivery vehicles without additional nano-carriers, have been considered as a novel approach for high efficient cancer therapy.39-42 In these systems, the drugloading capacities are significantly improved, even reaching 100%, which can improve drug accumulation in tumors and therapeutic efficacy.43, 44 Moreover, the carrier-free systems avoid biocompatibility issues associated with the carrier materials. Hitherto, two main strategies to make the drug self-delivery systems have been reported, namely prodrug self-delivery and pure drug self-delivery.45, 46 The prodrug self-delivery systems are obtained via covalent linking of drug molecules after careful pre-decoration with functional groups for further reaction.41, 47-55 However, the drugs might be inactivated during the decorating reactions. However, prodrug selfdelivery system only consists of pure drug, which is believed to be the ideal system for the desired therapeutic efficacy. The pure drug nano-systems can be realized by directly precipitating the drugs from soluble solutions using poor solvents, or obtaining the nanocrystals.56-58 A pioneering study of multi-drug delivery base on prodrug self-delivery has been reported by Huang et al. using amphiphilic drug-drug conjugates without any non-



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therapeutic components.44 The hydrophilic and hydrophobic units were covalently bonded for the self-assembly.59 Resultant nanoparticles exhibited longer retention time in the blood and higher accumulation in tumor tissues. Clearly, the drug-drug conjugate delivery systems require selection of the hydrophobic-hydrophilic drug pair.60 Furthermore, all the previous reports have mostly been dependent on molecular self-assembly via hydrophobic interactions, which means that drugs need to be rationally designed to be amphiphilic.61, 62 Unfortunately, many anticancer drugs are hydrophobic and do not have the intrinsic amphiphilicity required for self-assembly. Furthermore, the self-assembled drug self-delivery systems appear to be insufficiently stable to attack by endogenous biomolecules during drug transportation in vivo. A universal system, that is stable during blood circulation but rapidly releases drug after accumulation in tumor cells, still needs to be developed. Polyphosphazenes are a class of inorganic-organic hybrid polymers that are composed of alternating phosphorus and nitrogen atoms (–P=N–) as the backbone of the polymer chain, with two organic side groups attached to each phosphorus atom. Due to their intrinsic biodegradability, flexible molecular design capability and biocompatibility, polyphosphazenes have been widely applied in biological fields as bio-scaffolds,63, 64 bio-hydrogels delivery

carriers.69-72

However,

the

most

widely

reported

65-68

biologically

and drug available

polyphosphazenes have been linear polymers, prepared via ring-opening polymerization of hexachlorocyclotriphosphazene (HCCP) or living cationic polymerization under harsh conditions (250 °C in vacuum).73-76 Recently, our group has developed a facile method to prepare cyclomatrix polyphosphazene nanoparticles (CPPZs) for biological applications via direct precipitation polymerization of HCCP with di/multi-amino or di/multi-hydroxyl monomers


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under ambient conditions.77,

78

The CPPZs are pH-responsive due to hydrolysis of the

unsaturated P–N bonds to produce ammonium ions and phosphate groups.

Scheme 1. Schematic illustration of DSFDS: the DOX and HCCP are directly reacted and polymerized into CPPZ NPs. After circulation, the nanoparticles are delivered into tumor tissues via the EPR effect. The nanoparticles are taken up by lysosomes and then free drug is released for tumor therapy.

Since most anti-cancer drugs contain two or more amino/hydroxyl groups, they appear to be amenable to covalent linking with HCCP to form drug delivering nanoparticles. In this case, the drugs are not only loaded in the nanoparticles, but also act as foundational components of drug delivery carriers. In this paper, we report the construction of biodegradable cyclomatrix polyphosphazene nanoparticles through simple condensation polymerization of HCCP and doxorubicin (DOX), as illustrated in Scheme 1. The prepared nanoparticles were found to be 5 ACS Paragon Plus Environment


stable during circulation in the blood and were rapidly hydrolyzed in lysosomes after endocytosis. The nanoparticles therefore serve as a novel pH-responsive drug self-framed delivery system (DSFDS). Compared to traditional nano-carrier systems, the pH-sensitive DSFDs, or DOX-CPPZ NPs, have clear advantages in terms of synthesis, responsive release and drug-loading efficiency. Most importantly, this is a common method to construct DSFDS.

2. Materials and Methods 2.1. Materials: HCCP was purchased from TCI (Japan). DOX, acetonitrile (MeCN), and triethylamine (TEA) were obtained from Adamas-beta (China). LysoTracker Green, 4',6diamidino-2-phenylindole (DAPI) and the Annexin V-FITC/PI Cell Apoptosis Kit were purchased from Thermo Fisher. Cell culture medium (Dulbecco's Modified Eagle Medium with High Glucose, DMEM), penicillin/streptomycin and fetal bovine serum (FBS) were bought from Gibco (USA). Milli-Q water (Millipore) with resistivity of typically 18.2 MΩ·cm was applied in all experiments. The Cell Counting Kit-8 (CCK-8) was purchased from Beyotime Co. Ltd (China). HeLa and L929 cells were obtained from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science. 2.2. Synthesis of DOX-CPPZ NPs: HCCP (6 mg) and DOX (20 mg) were dissolved in 40 mL of MeCN, and then TEA (2 mL) was added as catalyst and acid-binding agent. The reaction was performed for 6 h at room temperature under ultrasonic irradiation (100 W, 40 kHz). After reaction, the resultant nanoparticles were obtained by centrifugation（5 min, 10280 rpm）and rinsed for three times with Milli-Q water. The DSFDS were then dried at room temperature under vacuum.


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The UV-vis absorbance of the supernatant solution at 480 nm was measured to check the unreacted DOX. The concentrations of unreacted DOX were then calculated under the guidance of a calibration curve with the same conditions. The loaded DOX in DSFDS could be calculated from the difference between the unreacted DOX and the totally feeding DOX. The formula is: RDOX-loaded = [(MDOX − Mun-DOX)/MDOX-CPPZ] × 100%，where MDOX-CPPZ is the drug-loading mass, MDOX is the total mass of DOX, and Mun-DOX is the unreacted DOX. 2.3. Characterization: The morphologies of resultant DSFDS were characterized by transmission electron microscopy (TEM, JEOL JEM-2100 at 200 kV, Japan) and field emission scanning electron microscope (FE SEM, FEI NONA-450 at 20 kV, USA). Image J was then used to measure the size and distribution of nanoparticles. Furthermore, the hydrated diameters of nanoparticles in aqueous medium were evaluated by dynamic light scatting using a Malvern Zetasizer Nano ZS at 633 nm. A spectrum 1000 (Perkin Elmer, USA) spectrometer was used to collect the Fourier-transform infrared (FT-IR) spectra. The chemical information of prepared DOX-CPPZs were also checked by ultraviolet-visible (UV-vis) absorption spectra carried out on a PE Lambda 35 spectrophotometer. For DOX quantification, the fluorescence spectra of DSFDS and solution were recorded on a Perkin-Elmer LS 50B fluorescence spectrometer. 2.4. In Vitro Drug Release: The in vitro drug release measurements were conducted by placing the DOX-CPPZ NPs (5 mg) into dialysis tubes (1 KD) with 100 mL phosphate-buffered saline (PBS) solution (pH 7.4 and 5.5) under gentle stirring at 37 °C. Then, the dialysate was collected and detected by UV spectrophotometer at 480 nm to real time determine the release of DOX. The corresponding standard curves for free DOX at 480 nm were pre-tested and presented in Figure S 2b and 2c.



2.5. Cell Viability Test: The bio-compatibility of DOX-CPPZ NPs was quantitatively assessed by a WST-8 assay. L929 or HeLa cells (200 µL, 1 × 105 cell mL-1) were seeded into a 96-well culture plate. The cells were incubated at 37 °C in a humidity atmosphere (5% CO2) for 12 h. Then, DOX-CPPZ NPs (0, 12.5, 25, 50, 100 and 200 µg mL-1 in FBS-free culture medium) were added into wells containing L929 cells at 37 °C for 4 h and then cultured in fresh media for 24, 48, and 72 h. Free DOX (0.25, 0.5, 1.0, and 2.0 µg mL-1 in FBS-free DMEM), DOX-CPPZ NPs (0.28, 0.56, 1.12, and 2.24 µg mL-1 in FBS-free DMEM) were added into wells containing HeLa cells at 37 °C for 4 h and then cultured in fresh media for 24, 48 and 72 h. Then, the WST8 assay was carried out to detect the cell viability. In detail, the optical absorbances of cells incubated with WST-8 at 450 nm were recorded by a microplate reader (Bio-rad, model 680). The viability was then calculated using pure cells as control. For more information about cell apoptosis, the cells were incubated with free DOX or DSFDS solution for 48 h, where the concentration of DOX was fixed at 2 µg mL-1. After that, cells were stained with Annexin V-FITC/PI according to the operation protocol. The fluorescence intensity was recorded on the flow cytometry (Becton-Dickinson FACS Calibur, USA). 2.6. In Vitro Uptake: HeLa cells were seeded into a four-chamber glass bottom dish. To observe endocytosis of nanoparticles by the cells, the HeLa cells were incubated with DOXCPPZ NPs for 4 h and then the cells were washed three times with PBS. Then, the cells incubated with fresh DMEM without FBS for further 1, 3, 6, 12 and 24 h. For staining, DMEM supplemented with LysoTracker Green was incubated with the cells for another 1 h and then rinsed with PBS (2 × 2 mL). Subsequently, the paraformaldehyde (4 wt %) was applied to fix cells for 30 min following by PBS rinsing for twice. At last, DAPI solution (0.1 µg mL-1) was used for staining the fixed cells for 15 min following by PBS rinsing for twice. A TCS SP8


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STED 3X super-resolution multiphoton confocal microscope (Leica, He−Ne and Ar lasers) was used to obtained fluorescence images of cells. 2.7. In Vivo Antitumor Activity Evaluation: All of the animal experiments were carried out strictly in according with the guide for the care and use of laboratory animals by Shanghai Jiao Tong University Animal Study Committee. Female BALB/c nude mice (4–6 weeks old) were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. The flank region of the mice was seeded with HeLa cells (1 × 107 in 100ml PBS) by subcutaneous injection. The volumes of the tumors were calculated as follow: Volume = Length × (Width)2/2, and the relative tumor volume (Vt/V0) was calculated by normalizing the volume to the initial size, where V0 was the tumor volume of beginning of therapy and Vt is the tumor volume of given time. 2.8. In Vivo Imaging and Biodistribution: The mice were randomly divided into three groups for different treatments: (a) PBS as control; (b) free DOX; (c) DOX-CPPZ NPs; (100µL, a DOX concentration of 0.17 mg mL-1, dosed intravenously via the tail vein). A Kodak multimode imaging system was applied for in vivo fluorescence imaging at different time points postinjection (Ex/Em at 488/590 nm). After 24h post-injection, the organs and the tumor were collected for fluorescence imaging and bio-distribution analysis. 2.9. In Vivo Tumor Growth Inhibition: Three groups ( n=5) of the HeLa tumors-bearing nude mice were randomly divided for different treatments: (a) PBS as control; (b) free DOX; (c) DOX-CPPZ NPs; (100 µL, a DOX concentration of 0.17 mg mL-1, dosed intravenously via the tail vein). The injections of different treatments were performed every four days for six times. The tumor change in volumes and body weight of mice were monitored at 1 day after every injection. At the end of the treatment, all of the mice were sacrificed. The organs and tumors were isolated from the mice for weighting and imaging. The organs were fixed in 4 wt %



paraformaldehyde for histology, hematoxylin-eosin (H&E) staining. The tumors were treated with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Statistical Analysis: Statistical analysis was performed using SPSS software by one-way ANOVA analysis. * p < 0.05, ** p < 0.01, *** p < 0.001.

3. Result and Discussion 3.1. Preparation and Characterization of DOX-CPPZ NPs In this study, the drug molecule (DOX) served as a monomer for polymerization into nanoparticles (Figure 1a). The hydroxyls of DOX acted as nucleophiles to substitute the chloride of HCCP in the presence of acid-binding agent (TEA), and formed cross-linked cyclomatrix polyphosphazene nanoparticles. The morphology of the nanoparticles was determined from SEM and TEM images (Figures 1b and 1c). The solid spheres, with an average diameter of approximately 125 nm, had the smooth surface that can be attributed to step-by-step chain growth from seed during condensation polymerization.69 The DLS analysis of an aqueous solution of DOX-CPPZ NPs indicated a hydrodynamic diameter of 133 nm with a narrow distribution range (polydispersity index = 0.119). The slightly larger size than that measured with SEM was due to the hydration layer around the nanoparticles. Photographs of DOX-CPPZ NPs suspension (inset in Figure 1d) indicated good dispersion stability.


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Figure 1. Preparation of DSFDs and characterization of DOX-CPPZ NPs. a) The reaction between HCCP and DOX; b) SEM image of DOX-CPPZ NPs; c) TEM image of DOX-CPPZ NPs; d) DLS data of DOX-CPPZ NPs (inset shows the optical photographs of free DOX and DOX-CPPZ suspension solutions); e) FTIR spectra of pure DOX, HCCP and DOX-CPPZ NPs; f) Energy dispersive spectroscopy spectrum of DOX-CPPZ NPs, the Silicon is introduced by the silicon substrate.

In addition, the chemical composition was checked by FT-IR (Figure 1e). The appearance of characteristic –P–O–Ar absorbance peaks at 970 cm−1 together with decreases of the signals at 3434 cm−1, due to DOX phenolic hydroxyl groups, and 1209 cm−1, corresponding to the P–Cl groups of HCCP, confirmed that DOX was covalently linked to the phosphazene molecule. The peak at 871 cm−1, corresponding to the P–N of HCCP, and the absorption peaks at 3331, 2933, 1730 and 1617–1480 cm−1 corresponding to the –NH2，C–H, C=O and anthraquinone ring of DOX, respectively, remained in the spectrum of DOX-CPPZ NPs. The combined results demonstrated the formation of polycondensate DOX-CPPZ NPs. However, the elemental



analysis from the EDX spectrum indicated that residual chlorine (Cl) atoms remained in the DOX-CPPZ NPs, which can be contributed to incomplete substitution as a result of steric hindrance (Figure 1f). The 31P NMR of HCCP and DOX-CPPZ NPs was further reported as shown in Figure S1. 31P NMR of -P- (Cl)2 in HCCP showed a characterized peak at 21.24 ppm. After cross-linking with DOX, a wide peak surrounding -8.22 ppm appeared, which was attributed to the P-(O-DOX)2 in the complicated chemical environment. In addition, a small peak at 15.74 ppm was assigned to Cl-P-(O-DOX) that also indicated an incomplete substitution. The drug-loading capacity is of primary importance in the evaluation of a drug delivery system.

A higher loading capacity not only decreases the required dose but also reduces

metabolic pressure on the kidneys and enhances therapeutic efficacy. In this study, the capacity of drug loaded in DSFDs was calculated based on the UV-vis spectrum. The pure DOX solution had a characteristic absorption peak at 480 nm (Figure S2a). After polymerization, the characteristic absorption peak shifted to 510 nm. The red shift of the peak indicated that the cross-linked HCCP rings played a role as auxochromic groups for the anthraquinone ring of DOX. The loading capacity of DOX was defined as the percentage of reacted DOX in the total DOX-CPPZ NPs. From a standard curve of DOX absorption at 480 nm, the loading capacity of DOX was calculated to be as high as 89.5% w/w. As illustrated in Scheme 1, one HCCP molecule could react with three DOX molecules (theoretically ≈ 87.1% w/w). The slightly higher actual drug-loading than theoretical could be attributed to physical adsorption of DOX in/on the drug self-framed nanoparticles. The drug-loading observed in this study is far higher than that of traditional drug delivery systems, which is the greatest advantage of drug self-framed delivery.


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Zeta potential analysis was applied to study the stability of DSDF systems. The Zeta potential of the DOX-CPPZ NPs in pH 7.4, 6.5 and 5.5 PBS were 24.3 mV±2.25 mV, 20.5 mV±3.35 mV, 10.4 mV±2.14 mV, respectively. That means that the DOX-CPPZ NPs are stability in neutral and slightly acidic condition and suitable for the drug delivery. 3.2. Cellular Uptake and Distribution

Figure 2. Confocal images of HeLa cells (blue: nuclei; green: lysosomes; red: DOX) after treatment with DOX-CPPZ NPs for 1, 3, 6, 12, and 24 h. The equivalent concentration of DOX was 2 µg mL-1. Scale bars=50 µm.

The cellular uptake of DOX-CPPZ NPs was determined using the immunofluorescence method imaged by confocal laser scanning microscopy. After incubated with DOX-CPPZ NPs for 4 h, HeLa cells were labeled simultaneously with DAPI for nuclei and Lyso-Tracker green 13 ACS Paragon Plus Environment


for lysosomes. Since the DOX present in DOX-CPPZ NPs still emits strong fluorescence at 590 nm when excited by laser at 488 nm (Figure S3), it can be used to track the distribution of DOX in HeLa cells. It can be observed that DOX-CPPZ NPs entered cells and were captured by lysosomes (Figure 2). No DOX signal was observed in nuclei at early time-points due to the shielding effect of nuclear membrane. However, after additional culture time, the acidic surrounding of the lysosomes provided mild conditions to facilitate degradation of the P–O bonds and release of DOX molecules. Then, free DOX permeated into cells cytoplasm and finally into nuclei after 12 h. This process was demonstrated by the decrease in the overlap coefficient of DOX fluorescence and LysoTracker green with culture time (Figure S4). The movement of DOX from lysosomes to nuclei could be contributed to pH-responsive release of DOX from DSFDs within lysosomes and subsequent net unidirectional diffusion from the cytoplasm across the nuclear membrane. In general, endocytosis is the primary pathway for entry of the drug delivery system or NPs into cells. The entrapped drug delivery system NPs are captured by lysosomes for degradation. Here, TEM was applied to check the location of the DSFDs after internalized in HeLa cells. It indicated that the majority of DOX-CPPZ NPs was localized in lysosomes (Figure S5). These experiments demonstrated that the DSFDs successfully crossed intracellular membranes via endocytosis, and delivered drugs into the lysosomes of cells. The pathway provides important information for the design and monitoring of DSFDs. 3.3. In Vitro Drug Release and Cytotoxicity of DOX-CPPZ NPs


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Figure 3. a) Release of DOX-CPPZ NPs at pH values of 7.4, 6.5 and 5.5 in vitro. b) Viability of L929 cells treated with various concentrations of DOX-CPPZ NPs for 24, 48, and 72 h. c) The killing efficiency of DOX-CPPZ NPs towards HeLa cells treated with various concentrations of DOX-CPPZ NPs for 24, 48, and 72 h. d) The killing efficiency of pure free-DOX towards HeLa cells treated with various concentrations of DOX for 24, 48, and 72 h. e) Flow cytometry analysis of HeLa cell (Q2-LR, Q2-UR, and Q2-UL) incubated with PBS, free DOX and DOXCPPZ NPs with fixed concentration of DOX at 2 µg mL-1) for 48 h. Q3-LR, necrotic cells; Q3UR, late apoptotic; Q3-UL, early apoptotic cells; Q3-LL, living cells.

The pH-dependent release behavior of DOX-CPPZ NPs was verified by dialysis in PBS at different pH values (7.4, 6.5 and 5.5) at 37 °C. As shown in Figure 3a, the release of DOX from DOX-CPPZ NPs in pH 7.4 PBS solution was at a low level (5%), which is beneficial for the stability of DOX-CPPZ NPs at physiological pH. At pH 6.6 and 5.5, DOX release sharply 15 ACS Paragon Plus Environment


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increased, indicating breakage of P–O bonds connecting DOX and phosphazene. In earlier reports, Allcock et al.79-84 demonstrated that linear polyphosphazene could be hydrolyzed in the presence of water, starting from cleavage of P–O and/or P–N bonds, followed by degradation of the hydroxylated polyphosphazene skeleton to phosphate and ammonium ions via further hydrolysis (illustrated in Figure S6). To understand the degradation process, the morphologies and diameters of nanoparticles at various conditions (with pH range from 7.4 to 4.0) were checked by TEM and DLS (Figure S7 and S8). It was obviously that the diameters decreased with the proceeding of degradation. The degradation process was greatly accelerated in an acidic environment. The DOX-CPPZ NPs therefore exhibited pH-responsiveness, a property that efficiently avoids premature drug release during blood circulation while enabling rapid release after uptake into tumor cells at the targeted sites. The pH-responsive release by DSFD is crucial for a drug delivery system. Meanwhile, it is also noted that the release percentage of DOX from the framework system is somewhat slow, in order to enhance the release rate, a study on the increase of degradation rate of the framework system in cells by introducing cystine into DOXCPPZ NPs is under way. For practical application, the cytotoxicity of DSFDs towards normal cells (using L929 as model cells) was evaluated in a WST-8 assay. The DSFDs exhibited good cytocompatibility over a wide range of concentrations (0–200 µg mL-1, Figure 3b). The slight decrease of L929 cell viability at 200 µg mL-1 DOX-CPPZ NPs might be due to physical effects of the nanoparticles. Furthermore, according to the degradation mechanism as mentioned above, the finally products of cyclomatrix polyphosphazene were phosphate and ammonium ions.

31

P NMR

analysis was also applied to check the degradation products of DOX-CPPZ NPs. As shown in Figure S9,

31

P NMR of final hydrolysis product of DOX-CPPZ NPs showed a signal peak at -


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0.18 ppm, which can be assigned phosphate root, indicating that DOX-CPPZ NPs became phosphate root after hydrolysis. Herein, the DSFDs were biocompatible. Subsequently, the therapeutic efficacy of the DSFDS was determined using free DOX as the control. It was found that both free DOX and DOX-CPPZ NPs had good cancer-killing ability (using HeLa cells as a model). The cell viabilities after treatment with DOX-CPPZ NPs (illustrated using 2 µg mL-1 DOX) were 76%, 66%, and 54% when cultured for 24, 48 and 72 h, respectively (Figure 3c). The different killing ability of DSFDS on normal cell L929 and cancer cell Hela should be attributed to the difference of the cellular membrane of two kinds of cells. It’s well known that cancer cells are mutant cells that can infinitely proliferate. The decrease of glycoproteins and other substances has taken place on the cellular membrane of cancer cells. Oncogenic RAS is believed to trigger micropinocytosis which is an endocytic process to internalize nutrient-containing fluids via cellular plasma membrane.85 Cancer cells have strong uptake capacity than normal cells. In other words, the penetrability of nanoparticles on cancer cell membrane was greatly improved. When fed with the same concentration of DSDF nanoparticles, there would be more nanoparticles that are uptaken into HeLa cells. Therefore, DSDF nanoparticles displayed good killing ability towards to HeLa cells, while presented good cytocompatibility towards normal L929 cells. These values were lower than those obtained with free DOX at the corresponding concentration and times (Figure 3d). Flow cytometry was employed to track the cell state after culture with 2 µg mL-1 of drug for 48 h (Figure 3e). Most cells that incubated with free DOX or DSFDS turned to be secondarily necrotic. The cellular apoptosis rate after treatment with DOXCPPZ NPs was lower because DOX was slowly released from the cross-linked polyphosphazene matrix, whereas the free drug was kept at a stable concentration during this evaluation. As



mentioned previously, the free anticancer drugs suffer from numerous problems, including low water solubility, short circulation time and low accumulation in tumors. Free DOX in vivo would distribute to other parts of the body via the circulation, therefore the concentration in tumor cells would be relatively low. Compared with free DOX, the DSFDs more readily accumulate in the tumor tissue ascribing to the EPR effect. More drugs would therefore be released in the tumor and a relatively good therapeutic effect should be achievable. This hypothesis was confirmed by the following in vivo experiment. 3.4. In Vivo Imaging and Biodistribution of DOX-CPPZ NPs To understand the distribution and metabolism of DSFDS, small-animal fluorescence imaging was applied to detect the real-time distribution of DOX in tumor-bearing nude mice. Figure 4a showed that intense fluorescence was observed throughout the whole body at 1 h after administration of either DOX or DOX-CPPZ NPs via the tail vein. Subsequently, free DOX began to be eliminated via metabolism (urine). Only a small amount of DOX remained in the tumor and liver after 12 h, and the fluorescence signal disappeared completely after 24 h. In contrast, contributing to EPR effect, DOX-CPPZ NPs displayed longer circulation time in blood and higher accumulation in the tumor. After 24 h, intense fluorescence was observed in the tumor, indicating good targeting of the drug delivery system.


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Figure 4. Fluorescence imaging for in vivo biodistribution of DOX-CPPZ NPs in tumor-bearing nude mice. a) Time-lapse fluorescence imaging in nude mice after intravenous administration of PBS, free DOX, and DOX-CPPZ NPs (from left to right). b) Fluorescence imaging of major organs and tumors after tail vein injection of PBS, pure DOX and DOX-CPPZ NPs for 24 h. c) Corresponding fluorescence intensity of major organs and tumors.

Ex vivo evaluation of excised organs was conducted to determine accumulation of DOX in vital tissues. Mice were randomly divided into three groups and treated with PBS, pure DOX or DOX-CPPZ NPs solutions respectively. After 24 h tail vein injection, the mice were then professionally dissected to obtain excised organs (Figure 4 b). There was a tendency for DOX to accumulate in heart, liver and kidney, but hardly any DOX remained in the tumor 24 h after



dosing with free drug. In contrast, a high concentration of DOX was observed in tumor tissues of mice treated with DOX-CPPZ NPs. These results demonstrated DSFDs reduced the drug release in blood circulation from DOX-CPPZ NPs and achieved acid-triggered DOX release in tumor cells after enhanced passive targeting. 3.5. In Vivo Biological Evaluation

Figure 5. Therapeutic evaluation of DOX-CPPZ NPs. a) The relative body weights of the mice (normalized to initial weight at day 0) were recorded for each treatment. b) The relative tumor volumes in HeLa tumor-bearing mice (normalized to initial weight at day 0) were recorded for each treatment. c) Images of excised tumors from mice via various treatments at the end of the experiment. d) Weights of excised tumors from mice via various treatments at the end of the experiment.


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HeLa tumor-bearing nude mice were used as a preclinical animal model to determine the in vivo antitumor efficacy of DOX-CPPZ NPs. The mice were injected with PBS, pure DOX, or DOX-CPPZ NPs (with fixed concentration of DOX at 0.17 mg mL-1) via tail vein (4 days apart) when the tumor size reached 200 mm3 in volume. In this study, relative body weights of each group of mice were measured to estimate toxicity during treatment. Mice treated with DOXCPPZ NPs or PBS exhibited a small increase of body weight, whereas there was a continuous and significant decrease in body weight in the free DOX group after each injection (Figure 5a). The results indicated that free DOX had serious side effects in Hela tumor-bearing nude mice, while DOX-CPPZ NPs had few relative impacts on the lives of mice such as diet. For more therapeutic information, the tumors in each group were isolated from treated mice for weighting and imaging. Compared to PBS, DOX-CPPZ NPs suppressed tumor growth by 70%, which was significantly better than the 15% inhibition by free DOX (Figures 5b–d). A TUNEL assay was also performed to study apoptosis of cells in tumor tissue. As shown in Figure 6a, DOX-CPPZ NPs displayed highest tumor killing ability, while free DOX exhibited unobvious differentiation compared to PBS. All of the results supported our hypothesis that the DSFD delivery system, with the EPR effect and long circulation time in blood, might have better therapeutic efficacy than free DOX, although the latter exhibited relatively high cytotoxicity in vitro. Potential side effects remain a big concern for clinical application of cancer therapeutics. The potential toxicity of the tested treatments towards key organs was further analyzed by H&E staining. H&E staining of major organs presented similar phenotypes indicating that there was no noticeable damage or obvious abnormality pathology between different treatments (Figure 6b). As mentioned earlier, the relative body weight (Figure 5a) decreased with tumor therapy, especially when using free DOX. Such a weight loss during free DOX therapy might be caused



by the heavy metabolic pressure on the kidney. Conversely, it has been demonstrated that DSFDs, with a longer circulation time and enhanced accumulation in tumor cells, can alleviate the acute side effects of DOX for cancer treatment in vivo.

Figure 6. a) Immunohistochemical images of dissected tumors from the different groups. Scale bars, 100 µm. b) H&E staining of the major organs after treatment with different therapeutic agents. Scale bars are 50 µm.

4. Conclusion In conclusion, we have exploited a facile and efficient method to synthesize a novel pHresponsive drug self-framed delivery system that has high drug-loading efficiency (~90 wt %), minimal side effects and high antitumor efficacy. The formation of the drug self-framed delivery system reduced the cytotoxicity of DOX, increased its blood circulation time, and increased the


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drug concentration in tumor tissue. Fluorescence images showed that the nanoparticles entered lysosomes by endocytosis. DOX molecules were released from the drug self-framed delivery system via hydrolysis in the lower pH environment of cells lysosomes, and then the released DOX, or part of it, located to the cell nucleus by diffusion. Animal experiments and histopathological sections demonstrated that the nanoparticles had low toxicity toward the main organs. Better outcomes for tumor suppression were achieved compared with free DOX due to the long circulation time in vivo and the EPR effect. Overall, the pH-responsive drug self-framed approach opens a new door for the design of drug delivery systems for cancer chemotherapy. Corresponding Author *Qinghua Lu: [email protected] Notes The authors declare no competing financial interest. Acknowledgements This work was supported by the National Science Foundation of China (51573089, 21704076). ASSOCIATED CONTENT Supporting Information This information is available free of charge via the Internet at http://pubs.acs.org/. Supporting information: S1.

31

P solid-state NMR of DOX-CPPZ NPs and HCCP; S2. UV−vis

spectra of free DOX, DOX-CPPZ NPs and the standard surve of the free DOX UV absorbance at 480 nm; S3. the fluorescent spectra of free DOX and DOX-CPPZ NPs; S4. the overlap coefficient of DOX fluorescence and LysoTracker Green based on the cell images; S5. the TEM images of the DOX-CPPZ NPs distribution in Hela cells; S6. the possible degradation mechanism of cyclomatrix

olyphosphazene; S7. TEM iamge of DOX-CPPZ NPs during



degradation; S8. DLS data of DOX-CPPZ NPs during degradation processes; S9.

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Biodegradable Cyclomatrix Polyphosphazene Nanoparticles: A Novel

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