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Novel pH-responsive biodegradable organosilica nanoparticles as drug delivery system for cancer therapy
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Colloids and Surfaces A journal homepage: www.elsevier.com/locate/colsurfa
Novel pH-responsive biodegradable organosilica nanoparticles as drug delivery system for cancer therapy ⁎
Shun Yang , Jie Fan, Shiting Lin, Yaru Wang, Chang Liu School of Chemistry and Materials Science, Jiangsu Normal University, Xuzhou, Jiangsu, 221116, China
G R A P H I C A L A B S T R A C T
Novel biodegradable hollow mesoporous organosilica nanoparticles (PBHMONs) based on pH-responsive silsesquioxane was successfully prepared for efficient anticancer drug delivery.
A R T I C LE I N FO
A B S T R A C T
Keywords: Biodegradable pH-responsive Organosilica Drug delivery
Although the biodegradability of silica has been verified, the complete and safe excretion of mesoporous silica nanoparticles (MSNs) from the biological system still impede their clinical translation. Herein, novel pH-responsive biodegradable hollow mesoporous organosilica nanoparticles (PBHMONs) based on pH-responsive silsesquioxane were successfully prepared for efficient anti-cancer drug delivery. pH-responsive cleavable bridges containing acetal moieties were prepared and then inserted directly into the framework of the MONs and the hollow nanostructures were achieved by silica-etching chemistry (alkaline etching). PBHMONs exhibited particularly high drug loading capacities for drugs (DOX) owing to the hollow mesoporous structure, and a uniquely low premature leakage owing to π-π interactions. Moreover, the pH-responsive biodegradation of the PBHMONs in the weakly acidic microenvironment of tumor cells led to partial drug release, as well as potential enabling rapid excretion and preventing long-term retention/toxicity.
1. Introduction Mesoporous silica nanoparticles (MSNs) are one of the most promising inorganic drug carriers and have attracted a great deal of attention in recent years owing to their unique structural properties, such as high surface area and pore volume, tunable pore size, and chemical
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stability. In addition, the surface of MSNs can be easily modified with functional groups for imaging, targeting, and other intelligent properties [1–10]. Hollow mesoporous silica nanoparticles (HMS) are a specific member of the MSNs nanofamily. The hollow core can act as a drug reservoir to improve the loading capacity, and the mesopores provide a controlled diffusion path for drug loading and release
Corresponding author. E-mail address: [email protected] (S. Yang).
https://doi.org/10.1016/j.colsurfa.2019.124133 Received 29 August 2019; Received in revised form 14 October 2019; Accepted 16 October 2019 Available online 20 October 2019 0927-7757/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Shun Yang, et al., Colloids and Surfaces A, https://doi.org/10.1016/j.colsurfa.2019.124133
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2. Experimental section
[11–13]. However, it remains difficult to ensure the complete and rapid excretion of silica NPs from biological systems despite the verified biodegradability of silica, which has impeded the clinical translation of MSNs [14,15]. In recent years, tumor microenvironment (TME)-responsive biodegradable mesoporous organosilica NPs (MONs) have attracted significant attention as anti-cancer drug nanocarriers owing to their enhanced degradability and low toxicity. MONs are organic-inorganic hybrid porous materials. The framework of MONs comprises an abundance of organic/inorganic bridges (silsesquioxane, O1.5Si-R-SiO1.5), which are homogeneously distributed throughout the framework at the molecular level. By adjusting the functional silsesquioxane in the MONs framework, the nature of the MONs can be easily altered [16–25]. Therefore, TME-responsive biodegradable MONs can be prepared by introducing TME-responsive cleavable bridges [26–29]. For example, intracellular bioreducing agent responsive-such as glutathione tripeptide-responsive-biodegradable MONs were synthesized by introducing disulfide bridges into the framework [30]. Chen et al. prepared biodegradable MONs with hollow structures using thioether bridges in the NP framework [20]. Oxamide-phenylene bridges were introduced to prepare trypsin-protein responsive biodegradable MONs [31]. These TME-responsive MONs could biodegrade in response to stimuli present in the intracellular environment once the target was reached, accelerating drug release. In addition to bioreducing agents and trypsin, pH has also been widely used as a stimulus for nanosystems for bio-applications owing to the much lower pH (e.g. 6.5–7.2 for extracellular tumor regions, pH 5–6.5 for endosome, and pH 4.5–5 for lysosomes) of the TME compared with normal tissue [32–36]. Accordingly, the design and fabrication of novel pH-responsive biodegradable MONs (PBHMONs) based on pH-responsive cleavable bridges would be an appealing way to promote the future clinical transformation of TMEresponsive MONs. In this work, we describe the synthesis of pH-responsive biodegradable hollow mesoporous organosilica NPs (PBHMONs). The pHresponsive cleavable bridges containing acetal moieties were prepared and then inserted directly into the MONs framework, and the hollow nanostructures were achieved following silica-etching chemistry (alkaline etching) during the synthesis process. As shown in Scheme 1, the hollow core of the PBHMONs provided a reservoir allowing a high drug loading capacity (Doxorubicin (DOX) was used as the model drug), and, in normal tissue, the drug release was delayed owing to the π-π interaction between DOX and organic components in the framework. Based on the enhanced permeability and retention (EPR) effect of tumor tissue, drug loaded PBHMONs would be expected to preferentially accumulate in tumor cells, where the weakly acidic microenvironment would stimulate the biodegradation of PBHMONs, leading to a greater tendency to release the loaded drug and rapid excretion.
2.1. Materials 1,4-Phthalaldehyde, p-toluenesulfonic acid (PTSA), and 2,2-bisbromomethyl-propane-1,3-diol were purchased from Energy Chemical Reagent Co. Ltd. Triethanolamine (TEOA), tetraethyl orthosilicate (TEOS), hexadecyl trimethyl ammonium chloride (CTAC, > 99.0%), and 3-aminopropyltriethoxysilane (APTES) were obtained from SigmaAldrich Co. (Shanghai, China). Other reagents were used as received. 2.2. Synthesis of pH-responsive compound 1 Compound 1 was synthesized in accordance with a previous report with some modifications [37]. In brief, p-toluenesulfonic acid (0.3 g), 1,4-phthalaldehyde (13 g), and 2,2-bis-bromomethyl-propane-1,3-diol (52 g) were dissolved in 200 mL of toluene and the solution was refluxed overnight. The water formed in the reaction was removed by an oil-water separator. The reaction mixture was then cooled to RT, filtered, and dried in vacuo at RT. The final product was a white powder. Yield: 90%. 1H NMR (400 MHz, DMSO-d6), d (ppm): 7.46 (s, 4H, C6H4), 5.50 (s, 2H, ArCH), 3.96–4.04 (12H, CCH2O and CCH2N), 3.46 (s, 4H, CCH2N). 2.3. Synthesis of pH-responsive biodegradable hollow mesoporous organosilica NPs (PBHMONs) Triethylamine (1.52 g, 15 mmol) and 3-aminopropyltriethoxysilane (3.32 g, 15 mmol) were added to a solution of compound 1 (0.933 g, 1.5 mmol) in THF (50 mL), and the mixture was stirred at 50 °C under N2 for 12 h. After cooling to RT, 20 mL of hexane was added and the ammonium salt was filtered through a short MgSO4 column. The clear solution was then concentrated and dried under vacuum at 60 °C overnight to obtain the pH-responsive silsesquioxane as a pale-yellow viscous oil. The silsesquioxane was used directly without any further treatment. PBHMONs was synthesized in accordance with a previous report with some modifications [38]. Typically, 2 g of CTAC were dissolved in 20 mL water and then mixed with 8 g of TEA aqueous solution (10 wt %). The solution was then stirred in an oil bath at 95 °C for 10 min before 1 mL of TEOS was added dropwise. The silica core (SiO2) was generated after 1 h. Then, 1 mL of mixed silicon sources (TEOS and different amounts of silsesquioxane) were added to the reaction system, which was left to react for another 4 h to achieve the SiO2@PBMONs core/shell structure. After centrifugation and washing with ethanol three times, the precipitate was dispersed in ethanol and stirred for 12 h at 78 °C three times to remove the CTAC, and then dispersed in ethanol (20 mL). Finally, 5 mL of SiO2@PBMONs solution was mixed with 100 mL of water containing 2.5 mL of ammonia solution and the mixture was stirred for 3 h at 95 °C. Hollow-structured pH-responsive
Scheme 1. Schematic representation of blood transport/tumor accumulation of PBHMONs and their pH-responsive biodegradation. 2
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biodegradable mesoporous organosilica NPs (PBHMONs) were finally collected by centrifugation and washed with water.
PBHMONs solutions for 2 h or 1 d. The culture media were removed and the cells were washed three times with PBS to remove non-internalized particles before analysis. The cells were observed using an inverted fluorescence microscope.
2.4. Biodegradation of PBHMONs PBHMONs (1 mg) were dispersed in phosphate buffered saline (PBS, 10 mL) at pH 6 and the suspension was sonicated for 10 min and then stirred at 37 °C. 1.5 mL aliquots were taken at different time points (from 0 to 5 d) for TEM analysis. The sample was then centrifuged and the supernatant was collected for dynamic light scattering (DLS) analysis.
2.8. In vitro cytotoxicity cytotoxicity A standard methyl thiazolyl tetrazolium (MTT, Sigma Aldrich) assay was used to assess the potential cytotoxicity of the as-fabricated PBHMONs towards KB cells. The study of therapeutic efficiency of DOXloaded PBHMONs was also based on KB cells and the viabilities of KB cells were determined by standard methyl thiazolyl tetrazolium (MTT, Sigma Aldrich) assay.
2.5. Drug loading and release Doxorubicin (DOX) was used as a model anticancer agent. Insoluble DOX was extracted from doxorubicin hydrochloride (DOX∙HCl) according to a previously reported procedure [39]. The DOX solution (5 mg/mL) was added to 0.8 mL of the as-prepared PBHMONs in tetrahydrofuran, followed by a slow addition of 10 mL of phosphate buffer (0.02 M, pH 7.4). The mixed solution was then shaken for 24 h to allow the diffusion of DOX into the NPs. Then, the drug-loaded nanoparticles were collected by centrifugation and washing with water/ethanol for 3 times. The concentrations of the remaining DOX solution and washing water/ethanol was determined using UV absorbance at 485 nm. The loading efficiency was calculated as follows: Loading efficiency = (C0V0−CtVt)/C0V0 where C0 and V0 are the concentration and volume of the added DOX, respectively, and Ct and Vt are the concentration and volume of free DOX after the centrifugation and washing, respectively. The drug release test was performed by suspending the DOX-loaded PBHMON nanocarriers in PBS buffers with different pH values. The mixed solution was shaken in a water bath at a constant temperature of 37 °C. To determine the amount of drug release at a particular time point, 1.0 mL of the solution was withdrawn after centrifugation and the same volume of PBS was introduced to maintain a constant volume. The drug concentration in the withdrawn solution was analyzed.
2.9. In vivo experiments experiments To develop the tumor model, KB cells (1 × 106 cells/site) were implanted subcutaneously into the legs of 4-week-old male athymic nude mice. Then mice were divided into three groups (n = 5 per group) for various treatments: (1) Saline; (2) PBHMONs; (3) DOX-loaded PBHMONs (once every 2 d at 10 mg/mL). The tumor size were measured by a caliper every other day and calculated as the volume= (tumor length)×(tumor width)2/2. And the relative tumor volumes were calculated as V/V0 (V0 was the initial tumor volume). All animal procedures were performed in accordance with the internationally accepted principles and Guidelines for the Care and Use of Laboratory Animals of Jiangsu Normal University and the experiment protocols were approved by the Institutional Animal Ethical Committee of the Jiangsu Normal University. All efforts were made to minimize suffering. 3. Results and discussion The pH-responsive organic molecule (compound 1), containing acetal moieties, was synthesized using the procedure shown in Scheme S1. The structure of the molecule was determined by 1H NMR and the findings are given in the experimental section. To demonstrate the cleavage of the acetal moieties under acidic conditions, a given amount of compound 1 was dissolved in dichloromethane and the solution was mixed with 20 mL of PBS at pH 6. After stirring for 2 h, the organic phase was collected and the dichloromethane was evaporated. The residue was evaluated using 1H NMR and the spectrum is shown in Fig. S1. Two new aromatic signals at δ = 8.15 and 3.06∼3.47 ppm appeared in the 1H NMR spectrum and were shown to be from 1,4Phthalaldehyde and 2,2-Bis(bromomethyl)propane-1,3-diol, respectively. This finding supports the cleavability of compound 1, which is key to the biodegradation of PBHMONs. To prepare pH-responsive biodegradable hollow mesoporous organosilica NPs (PBHMONs), pH-responsive silsesquioxane was first synthesized based on compound 1 and 3-aminopropyltriethoxysilane, and given amounts of the product without further treatment were mixed directly with TEOS to form the mixed silicon sources. The SiO2@
2.6. Cell culture and preparation Human epidermoid carcinoma cells (KB cells) (purchased from Shanghai Cell Institute Country Cell Bank, China) were cultured at 37 °C in a humidified incubator (5% CO2 in air, v/v) as a monolayer in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum. 2.7. Cellular uptake of DOX-loaded PBHMONs KB cells were seeded in 96-well plates (1.3 × 104 cells/well) and incubated overnight at 37 °C in a humidified incubator. The dispersion of DOX-loaded PBHMONs was prepared in RPMI-1640 medium. The DOX concentration in the PBHMONs nanocarriers was 10 μg/mL. Cells were washed twice with PBS and incubated with the above DOX-loaded
Scheme 2. Synthetic scheme for PBHMONs. 3
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Fig. 1. SEM (a, b) and TEM (c, d) images of PBHMONs based on a Si source molar ratio of 50:50.
clearly show that the PBHMONs were broken down in the acidic environment and exhibited significant biodegradation after 5 d, which was faster than previous reported redox responsive mesoporous organosilica nanoparticles containing disulfide (S-S) bridges (7 d). [30,40] The SEM image (Fig. 4f) of PBHMONs after treatment with acid for 5 d also confirms the degradation of the NPs. For comparison, PBHMONs (0.1 mg/mL) were also stirred at 37 °C in PBS (pH = 7.4, up to 5 d), and the TEM images after 5 d are shown in Fig. S4. In addition, small fragments were present in the supernatant due to the degradation of the PBHMONs exposed to the acidic environment. The supernatant was further analyzed by dynamic light scattering (DLS) after centrifugation (Fig. 4g). The results supported the expectation that the as prepared PBHMONs would biodegrade faster in acidic tumor tissues, leading to rapid excretion and contributing significantly to the potential clinical translation of the system. To study the cargo loading performance of the PBHMON nanocarriers, Doxorubicin (DOX) was used as a model drug. The concentration of DOX was determined by UV–vis spectroscopy and the loading efficiency and capacity were calculated to be 86.4% and 162.9 ug-DOX∙mg/nanocarriers, respectively. To our knowledge, the drug loading efficiency of PBHMONs was much higher than those of traditional drug delivery systems (MSNs and organic micelles) [39]. In addition, the collected DOX-loaded PBHMONs was washed with PBS and almost no DOX could be detected in the washing water, indicating that most of the drug was loaded in the hollows, mesopores and inner surface of shells, instead of attached on the outer surface of shells. The biodegradation behavior and structure evolution enhanced the release properties of the PBHMONs nanoparticles. DOX-loaded PBHMONs were prepared and their release behavior was investigated using PBS solutions with different pH (pH 6.0 and 7.4, simulating tumor and normal cells) at 37 °C. As shown in Fig. 5, ∼80% of the DOX was released from the PBHMONs at pH 6.0 while only ∼20% of the DOX was released in a neutral environment after 150 h. The π-π interaction
PBMONs core/shell structure with a silica core was synthesized via the Stöber method, and PBHMONs were obtained after an etching process (Scheme 2). The molar ratio of TEOS and pH-responsive silsesquioxane affected the morphology of the bulk organosilica nanoparticles. Three different ratios (70:30, 50:50, and 30:70, TEOS:pH-responsive silsesquioxane, based on the Si source) were used to prepare PBHMONs, and the structures were analyzed by TEM. As shown in Fig. S2, the shells of the PBHMONs based on the 70:30 mol ratio were too thick, and the shells of PBHMONs based on the 30:70 mol ratio were broken after the etching process. The morphology of PBHMONs based on a molar ratio of 50:50 can be observed in Fig. 1, both the SEM and TEM images show the homogenous spherical morphology of the PBHMONs evidencing a good degree of particles size monodispersity (∼100 nm), and the hollow structure can be clearly observed from the TEM images. Dynamic light scattering (DLS) measurements were used to evaluate the particle size, and the results (Fig. S3) indicated that the diameter of the PBHMONs was ∼100 nm, which was in accordance with the findings from the microscopy images. The specific area and pore size were measured by N2 adsorption analysis. The results shown in Fig. 2 shows that the PBHMONs had a surface area of 862.5 m2/g with an average pore size of 2.2 nm. The large surface area is beneficial for drug loading and the mesopores provide a controlled diffusion path for drug payloads. The presence of organic bridges, which affect the nature of the overall structure and control the pH-responsive biodegradation of PBHMONs, was determined by energy dispersive spectrometry (EDS), and the spectrum is shown in Fig. 3. The presence of nitrogen (N) strongly supports the presence of pH-responsive organic groups in the shell of the as prepared particles. To study the structure breakdown of PBHMONs, PBHMONs (0.1 mg/mL) were stirred in acidic PBS at 37 °C (pH = 6, up to 5 days), and aliquots of the suspension were collected at regular intervals, and analyzed by TEM observation (Fig. 4). TEM images of the suspension 4
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Fig. 2. (a) N2 adsorption-desorption isotherms and (b) the corresponding pore-size distributions for PBHMONs.
Fig. 3. EDS analysis of the PBHMONs nanoparticles.
Fig. 4. Characterization of the biodegradation of PBHMONs. (a–e) TEM analysis of a suspension of PBHMONs (0.1 mg/mL, PBS, pH = 6, 37 °C) after 0, 0.5, 1, 3, and 5 d, (f) SEM images of a suspension of PBHMONs (0.1 mg/mL, PBS, pH = 6, 37 °C) after 5 d, (g) DLS analysis of the supernatant of the centrifuged dispersion of PBHMONs (0.1 mg/mL, PBS, pH = 6, 37 °C) after 1, 3, and 5 d.
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mainly distributed in cytoplasm at 2 h and nucleus at 1d, confirming the DOX leakage form the nanocarriers. The cytotoxicity of free PBHMONs was studied by MTT assay. As shown in Fig. 7a, KB cells maintained a high cell viability (> 80%) after being incubated with PBHMONs for 48 h, and the cytotoxicity of the NPs remained low when the concentration was increased. In addition, the fragments of PBHMONs that formed after acid treatment for 5 days were collected, and the cytotoxicity also remained low. These findings clearly demonstrate that both the free and degraded PBHMONs were biocompatible and nontoxic, which is important for their further biomedical application. The biodegradation of the PBHMONs and the efficient leakage of the loaded drug molecules enhanced the cytotoxicity of the DOX-loaded PBHMONs in vitro. KB cells were incubated with DOX-loaded HMS, DOX-loaded PBHMONs, and free DOX (5, 10, 20 and 40 ug-DOX/mL) for 24 h and the cell viability evaluated using MTT is shown in Fig. 7b. Compared with the non-biodegradable HMS particles loaded with DOX, the pH-responsive biodegradation of PBHMONs caused by the acid endo/lysosomes led to more efficient DOX release and subsequent diffusion of DOX from the cytoplasm to the nucleus, and it is well-known that the ultimate target of DOX is DNA to form DNA-DOX adduct to inhibit transcription and induce the death of tumor cells, which resulted in a clear difference in cytotoxicity between the DOX@HMS nanocarriers and DOX-loaded PBHMONs nanocarriers. In addition, KB cells were also incubated with pure PBHMONs for contrast, obviously high cell viability indicated that apoptosis or necrosis was not triggered by the nanoparticles in the cancer cells. Finally, TUNEL assay was carried out to demonstrate whether the above mentioned pH-responsive biodegradation of PBHMONs may result in enhancement of cell apoptosis. As shown Fig. S7, cells incubated with DOX-loaded PBHMONs have a higher apoptosis rate, which was consistent with the MTT results. To study the antitumor efficiency of DOX-loaded PBHMONs, athymic nude mice bearing KB cells were randomized into 3 treatment groups and intratumorally (i.t.) injected with ca. 50 μL of saline, PBHMONs, or DOX-loaded PBHMONs (10 mg /mL). The tumor volumes and body weights were measured over the subsequent 16 d. As shown in Fig. 8a and Fig. 8b, the tumor volume of the PBHMONs group exhibited almost no difference to the saline group, confirming that PBHMONs showed no efficient inhibition of tumor growth. In contrast, effective elimination of tumors was achieved in the group of mice treated with DOX-loaded PBHMONs owing to the effective drug release following pH responsive biodegradation. The negligible variation in the average weights of the different groups (Fig. S8) indicated that the biodegradation of PBHMONs did not affect the health of the mice. The mice were euthanized at day 16 and the major organs were
Fig. 5. DOX release profiles of DOX-loaded PBHMONs at pH = 6.0 and 7.4, and DOX-loaded HMS at pH = 6. Error bars indicate mean ± SD, n = 3.
between DOX and the pH-responsive bridges is expected to delay the drug release in normal organs/tissues, while the biodegradation of the NPs in tumor tissue facilitates the drug release; leading to relatively high biosafety, which is important for further clinical translation and application in cancer treatment. Moreover, HMS with a diameter of 140 nm (Fig. S5) were synthesized according to our previous work and loaded with DOX [41]. And the release behavior DOX-loaded HMS at pH 6.0 was also recorded, the lower release efficiency of DOX-loaded HMS demonstrated that the degradation of PBHMONs could accelerate the release of loaded DOX molecules. Owing to the red fluorescence of DOX, the intracellular activity of the DOX-loaded PBHMONs could be monitored without any additional modification. KB cells were first incubated with DOX-loaded PBHMONs in which the DOX concentration was calculated to be 10 μg mL−1. Following the removal of non-internalized NPs, cells were stained with DAPI, and then the red fluorescence of the DOX inside the cells was monitored using an inverted fluorescence microscope over periods of 2 h and 1 d. As shown in Fig. 6, the KB cells were able to take up the drug nanocarriers via an endocytosis pathway, and it was observed that DOX distributed throughout the whole cell after 2 h, while it was mainly found in the nucleus after 1 d. This observation was primarily the result of DOX leakage. To further demonstrate the distribution of DOX, co-localization fluorescence measurements was carried out using magnified cell images. As shown in Fig. S6, the fluorescence of DOX was
Fig. 6. Fluorescence images of KB cells incubated with DOX-loaded PBHMONs for 2 h (top) and 1 d (bottom). 6
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Fig. 7. Viability of KB cells after incubation with (a) PBHMONs for 48 h, and (b) DOX-loaded HMS, DOX-loaded PBHMONs, free DOX and PBHMONs for 24 h. Error bars indicate mean ± SD, n = 3. **P < 0.01, ***P < 0.001. Fig. 8. In vivo treatment with DOX-loaded PBHMONs (a) Tumor growth curves of mice after various treatments. (b) Image of tumor tissues dissected after treatment. (c) Survival curves of mice after various treatments. (d) H& E stained major organs collected from different treatment groups. Error bars indicate mean ± SD, n = 5. ***P < 0.001.
that could be stimulated by weakly acidic environments were designed and fabricated. The hollow structure of the prepared PBHMONs gave the nanocarriers a high drug loading capacity, while the use of DOX as a model drug, and the π-π interactions between the introduced pH-responsive groups in the shell and the DOX molecules, prevented the release of the drug in normal tissues. Once the DOX-loaded PBHMONs entered the tumor cells via EPR, the weakly acidic microenvironment induced the biodegradation of the PBHMONs, leading to partial drug release upon degradation of the carrier and efficient cancer cell killing, as well as rapid excretion. This study paves the way for the development of the next generation of hybrid nanocarriers tailored for cargo delivery into tumor cells for disease-targeted treatments
collected then H&E stained. As shown in Fig.8d, there was no apparent difference between the saline group, and the PBHMONs and DOXloaded PBHMONs groups, in the heart, liver, spleen, lung, and kidney, confirming that the synthesized PBHMONs caused no significant damage to the major organs. In addition, the survival rates of three further groups of athymic nude mice bearing KB cells treated using the same strategy were recorded. A lower survival rate was observed in the control groups owing to the fast growth of the tumors (Fig. 8c), demonstrating the drug delivery efficiency of PBHMONs.
4. Conclusions In summary, novel hollow tumor microenvironment (TME)-responsive biodegradable mesoporous organosilica nanoparticles (MONs) 7
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Declaration of Competing Interest
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We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled “Novel pH-responsive Biodegradable Organosilica Nanoparticles as Drug Delivery System for Cancer Therapy”. Acknowledgements We gratefully acknowledge the financial support provided by National Natural Science Foundation of China (51802127), Natural Science Foundation of Jiangsu Province (BK20170239), the Priority Academic Program Development of Jiangsu Higher Education Institutions and Jiangsu Normal University scientific research fund (16XLR005). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfa.2019.124133. References [1] W.S. Lin, W.Y. Huang, X.D. Zhou, Y.F. Ma, Toxicol. Appl. Pharmacol. 217 (2006) 252. [2] R.K. Kankala, C.G. Liu, A.Z. Chen, S.B. Wang, P.Y. Xu, L.K. Mende, C.L. Liu, C.H. Lee, Y.F. Hu, ACS Biomater. Sci. Eng. 3 (2017) 2431–2442. [3] R.K. Kankala, H. Zhang, C.G. Liu, K.R. Kanubaddi, C.H. Lee, S.B. Wang, W. Cui, H.A. Santos, K.L. Lin, A.Z. Chen, Adv. Funct. Mater. (2019) 1902652. [4] L. Tang, T.M. Fan, L.B. Borst, J. Cheng, ACS Nano 6 (2012) 3954. [5] A. Agostini, L. Mondragon, A. Bernardos, R. Martinez-Manez, M.D. Marcos, F. Sancenon, J. Soto, A. Costero, C. Manguan-Garcia, R. Perona, M. Moreno-Torres, R. Aparicio-Sanchis, J. Ramon Murguia, Angew. Chem. Int. Ed. 51 (2012) 10556. [6] H. Mekaru, J. Lu, F. Tamanoi, Adv. Drug Del. Rev. 95 (2015) 40. [7] C. Coll, L. Mondragon, R. Martinez-Manez, F. Sancenon, M. Dolores Marcos, J. Soto, P. Amoros, E. Perez-Paya, Angew. Chem. Int. Ed. 50 (2011) 2138. [8] C.G. Liu, Y.H. Han, J.T. Zhang, R.K. Kankala, S.B. Wang, A.Z. Chen, Chem. Eng. J. 370 (2019) 1188–1199. [9] K.C. Kao, C.Y. Mou, Microporous Mesoporous Mater. 169 (2013) 7. [10] N.Ž. Knežević, J.O. Durand, Nanoscale 7 (2015) 2199. [11] W.R. Zhao, M.D. Lang, Y.S. Li, L. Li, J.L. Shi, J. Mater. Chem. 19 (2009) 2778. [12] Y. Chen, H.R. Chen, L.M. Guo, Q.J. He, F. Chen, J. Zhou, J.W. Feng, J.L. Shi, ACS Nano 4 (2010) 529.
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Colloids and Surfaces A journal homepage: www.elsevier.com/locate/colsurfa
Novel pH-responsive biodegradable organosilica nanoparticles as drug delivery system for cancer therapy ⁎
Shun Yang , Jie Fan, Shiting Lin, Yaru Wang, Chang Liu School of Chemistry and Materials Science, Jiangsu Normal University, Xuzhou, Jiangsu, 221116, China
G R A P H I C A L A B S T R A C T
Novel biodegradable hollow mesoporous organosilica nanoparticles (PBHMONs) based on pH-responsive silsesquioxane was successfully prepared for efficient anticancer drug delivery.
A R T I C LE I N FO
A B S T R A C T
Keywords: Biodegradable pH-responsive Organosilica Drug delivery
Although the biodegradability of silica has been verified, the complete and safe excretion of mesoporous silica nanoparticles (MSNs) from the biological system still impede their clinical translation. Herein, novel pH-responsive biodegradable hollow mesoporous organosilica nanoparticles (PBHMONs) based on pH-responsive silsesquioxane were successfully prepared for efficient anti-cancer drug delivery. pH-responsive cleavable bridges containing acetal moieties were prepared and then inserted directly into the framework of the MONs and the hollow nanostructures were achieved by silica-etching chemistry (alkaline etching). PBHMONs exhibited particularly high drug loading capacities for drugs (DOX) owing to the hollow mesoporous structure, and a uniquely low premature leakage owing to π-π interactions. Moreover, the pH-responsive biodegradation of the PBHMONs in the weakly acidic microenvironment of tumor cells led to partial drug release, as well as potential enabling rapid excretion and preventing long-term retention/toxicity.
1. Introduction Mesoporous silica nanoparticles (MSNs) are one of the most promising inorganic drug carriers and have attracted a great deal of attention in recent years owing to their unique structural properties, such as high surface area and pore volume, tunable pore size, and chemical
⁎
stability. In addition, the surface of MSNs can be easily modified with functional groups for imaging, targeting, and other intelligent properties [1–10]. Hollow mesoporous silica nanoparticles (HMS) are a specific member of the MSNs nanofamily. The hollow core can act as a drug reservoir to improve the loading capacity, and the mesopores provide a controlled diffusion path for drug loading and release
Corresponding author. E-mail address: [email protected] (S. Yang).
https://doi.org/10.1016/j.colsurfa.2019.124133 Received 29 August 2019; Received in revised form 14 October 2019; Accepted 16 October 2019 Available online 20 October 2019 0927-7757/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Shun Yang, et al., Colloids and Surfaces A, https://doi.org/10.1016/j.colsurfa.2019.124133
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2. Experimental section
[11–13]. However, it remains difficult to ensure the complete and rapid excretion of silica NPs from biological systems despite the verified biodegradability of silica, which has impeded the clinical translation of MSNs [14,15]. In recent years, tumor microenvironment (TME)-responsive biodegradable mesoporous organosilica NPs (MONs) have attracted significant attention as anti-cancer drug nanocarriers owing to their enhanced degradability and low toxicity. MONs are organic-inorganic hybrid porous materials. The framework of MONs comprises an abundance of organic/inorganic bridges (silsesquioxane, O1.5Si-R-SiO1.5), which are homogeneously distributed throughout the framework at the molecular level. By adjusting the functional silsesquioxane in the MONs framework, the nature of the MONs can be easily altered [16–25]. Therefore, TME-responsive biodegradable MONs can be prepared by introducing TME-responsive cleavable bridges [26–29]. For example, intracellular bioreducing agent responsive-such as glutathione tripeptide-responsive-biodegradable MONs were synthesized by introducing disulfide bridges into the framework [30]. Chen et al. prepared biodegradable MONs with hollow structures using thioether bridges in the NP framework [20]. Oxamide-phenylene bridges were introduced to prepare trypsin-protein responsive biodegradable MONs [31]. These TME-responsive MONs could biodegrade in response to stimuli present in the intracellular environment once the target was reached, accelerating drug release. In addition to bioreducing agents and trypsin, pH has also been widely used as a stimulus for nanosystems for bio-applications owing to the much lower pH (e.g. 6.5–7.2 for extracellular tumor regions, pH 5–6.5 for endosome, and pH 4.5–5 for lysosomes) of the TME compared with normal tissue [32–36]. Accordingly, the design and fabrication of novel pH-responsive biodegradable MONs (PBHMONs) based on pH-responsive cleavable bridges would be an appealing way to promote the future clinical transformation of TMEresponsive MONs. In this work, we describe the synthesis of pH-responsive biodegradable hollow mesoporous organosilica NPs (PBHMONs). The pHresponsive cleavable bridges containing acetal moieties were prepared and then inserted directly into the MONs framework, and the hollow nanostructures were achieved following silica-etching chemistry (alkaline etching) during the synthesis process. As shown in Scheme 1, the hollow core of the PBHMONs provided a reservoir allowing a high drug loading capacity (Doxorubicin (DOX) was used as the model drug), and, in normal tissue, the drug release was delayed owing to the π-π interaction between DOX and organic components in the framework. Based on the enhanced permeability and retention (EPR) effect of tumor tissue, drug loaded PBHMONs would be expected to preferentially accumulate in tumor cells, where the weakly acidic microenvironment would stimulate the biodegradation of PBHMONs, leading to a greater tendency to release the loaded drug and rapid excretion.
2.1. Materials 1,4-Phthalaldehyde, p-toluenesulfonic acid (PTSA), and 2,2-bisbromomethyl-propane-1,3-diol were purchased from Energy Chemical Reagent Co. Ltd. Triethanolamine (TEOA), tetraethyl orthosilicate (TEOS), hexadecyl trimethyl ammonium chloride (CTAC, > 99.0%), and 3-aminopropyltriethoxysilane (APTES) were obtained from SigmaAldrich Co. (Shanghai, China). Other reagents were used as received. 2.2. Synthesis of pH-responsive compound 1 Compound 1 was synthesized in accordance with a previous report with some modifications [37]. In brief, p-toluenesulfonic acid (0.3 g), 1,4-phthalaldehyde (13 g), and 2,2-bis-bromomethyl-propane-1,3-diol (52 g) were dissolved in 200 mL of toluene and the solution was refluxed overnight. The water formed in the reaction was removed by an oil-water separator. The reaction mixture was then cooled to RT, filtered, and dried in vacuo at RT. The final product was a white powder. Yield: 90%. 1H NMR (400 MHz, DMSO-d6), d (ppm): 7.46 (s, 4H, C6H4), 5.50 (s, 2H, ArCH), 3.96–4.04 (12H, CCH2O and CCH2N), 3.46 (s, 4H, CCH2N). 2.3. Synthesis of pH-responsive biodegradable hollow mesoporous organosilica NPs (PBHMONs) Triethylamine (1.52 g, 15 mmol) and 3-aminopropyltriethoxysilane (3.32 g, 15 mmol) were added to a solution of compound 1 (0.933 g, 1.5 mmol) in THF (50 mL), and the mixture was stirred at 50 °C under N2 for 12 h. After cooling to RT, 20 mL of hexane was added and the ammonium salt was filtered through a short MgSO4 column. The clear solution was then concentrated and dried under vacuum at 60 °C overnight to obtain the pH-responsive silsesquioxane as a pale-yellow viscous oil. The silsesquioxane was used directly without any further treatment. PBHMONs was synthesized in accordance with a previous report with some modifications [38]. Typically, 2 g of CTAC were dissolved in 20 mL water and then mixed with 8 g of TEA aqueous solution (10 wt %). The solution was then stirred in an oil bath at 95 °C for 10 min before 1 mL of TEOS was added dropwise. The silica core (SiO2) was generated after 1 h. Then, 1 mL of mixed silicon sources (TEOS and different amounts of silsesquioxane) were added to the reaction system, which was left to react for another 4 h to achieve the SiO2@PBMONs core/shell structure. After centrifugation and washing with ethanol three times, the precipitate was dispersed in ethanol and stirred for 12 h at 78 °C three times to remove the CTAC, and then dispersed in ethanol (20 mL). Finally, 5 mL of SiO2@PBMONs solution was mixed with 100 mL of water containing 2.5 mL of ammonia solution and the mixture was stirred for 3 h at 95 °C. Hollow-structured pH-responsive
Scheme 1. Schematic representation of blood transport/tumor accumulation of PBHMONs and their pH-responsive biodegradation. 2
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biodegradable mesoporous organosilica NPs (PBHMONs) were finally collected by centrifugation and washed with water.
PBHMONs solutions for 2 h or 1 d. The culture media were removed and the cells were washed three times with PBS to remove non-internalized particles before analysis. The cells were observed using an inverted fluorescence microscope.
2.4. Biodegradation of PBHMONs PBHMONs (1 mg) were dispersed in phosphate buffered saline (PBS, 10 mL) at pH 6 and the suspension was sonicated for 10 min and then stirred at 37 °C. 1.5 mL aliquots were taken at different time points (from 0 to 5 d) for TEM analysis. The sample was then centrifuged and the supernatant was collected for dynamic light scattering (DLS) analysis.
2.8. In vitro cytotoxicity cytotoxicity A standard methyl thiazolyl tetrazolium (MTT, Sigma Aldrich) assay was used to assess the potential cytotoxicity of the as-fabricated PBHMONs towards KB cells. The study of therapeutic efficiency of DOXloaded PBHMONs was also based on KB cells and the viabilities of KB cells were determined by standard methyl thiazolyl tetrazolium (MTT, Sigma Aldrich) assay.
2.5. Drug loading and release Doxorubicin (DOX) was used as a model anticancer agent. Insoluble DOX was extracted from doxorubicin hydrochloride (DOX∙HCl) according to a previously reported procedure [39]. The DOX solution (5 mg/mL) was added to 0.8 mL of the as-prepared PBHMONs in tetrahydrofuran, followed by a slow addition of 10 mL of phosphate buffer (0.02 M, pH 7.4). The mixed solution was then shaken for 24 h to allow the diffusion of DOX into the NPs. Then, the drug-loaded nanoparticles were collected by centrifugation and washing with water/ethanol for 3 times. The concentrations of the remaining DOX solution and washing water/ethanol was determined using UV absorbance at 485 nm. The loading efficiency was calculated as follows: Loading efficiency = (C0V0−CtVt)/C0V0 where C0 and V0 are the concentration and volume of the added DOX, respectively, and Ct and Vt are the concentration and volume of free DOX after the centrifugation and washing, respectively. The drug release test was performed by suspending the DOX-loaded PBHMON nanocarriers in PBS buffers with different pH values. The mixed solution was shaken in a water bath at a constant temperature of 37 °C. To determine the amount of drug release at a particular time point, 1.0 mL of the solution was withdrawn after centrifugation and the same volume of PBS was introduced to maintain a constant volume. The drug concentration in the withdrawn solution was analyzed.
2.9. In vivo experiments experiments To develop the tumor model, KB cells (1 × 106 cells/site) were implanted subcutaneously into the legs of 4-week-old male athymic nude mice. Then mice were divided into three groups (n = 5 per group) for various treatments: (1) Saline; (2) PBHMONs; (3) DOX-loaded PBHMONs (once every 2 d at 10 mg/mL). The tumor size were measured by a caliper every other day and calculated as the volume= (tumor length)×(tumor width)2/2. And the relative tumor volumes were calculated as V/V0 (V0 was the initial tumor volume). All animal procedures were performed in accordance with the internationally accepted principles and Guidelines for the Care and Use of Laboratory Animals of Jiangsu Normal University and the experiment protocols were approved by the Institutional Animal Ethical Committee of the Jiangsu Normal University. All efforts were made to minimize suffering. 3. Results and discussion The pH-responsive organic molecule (compound 1), containing acetal moieties, was synthesized using the procedure shown in Scheme S1. The structure of the molecule was determined by 1H NMR and the findings are given in the experimental section. To demonstrate the cleavage of the acetal moieties under acidic conditions, a given amount of compound 1 was dissolved in dichloromethane and the solution was mixed with 20 mL of PBS at pH 6. After stirring for 2 h, the organic phase was collected and the dichloromethane was evaporated. The residue was evaluated using 1H NMR and the spectrum is shown in Fig. S1. Two new aromatic signals at δ = 8.15 and 3.06∼3.47 ppm appeared in the 1H NMR spectrum and were shown to be from 1,4Phthalaldehyde and 2,2-Bis(bromomethyl)propane-1,3-diol, respectively. This finding supports the cleavability of compound 1, which is key to the biodegradation of PBHMONs. To prepare pH-responsive biodegradable hollow mesoporous organosilica NPs (PBHMONs), pH-responsive silsesquioxane was first synthesized based on compound 1 and 3-aminopropyltriethoxysilane, and given amounts of the product without further treatment were mixed directly with TEOS to form the mixed silicon sources. The SiO2@
2.6. Cell culture and preparation Human epidermoid carcinoma cells (KB cells) (purchased from Shanghai Cell Institute Country Cell Bank, China) were cultured at 37 °C in a humidified incubator (5% CO2 in air, v/v) as a monolayer in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum. 2.7. Cellular uptake of DOX-loaded PBHMONs KB cells were seeded in 96-well plates (1.3 × 104 cells/well) and incubated overnight at 37 °C in a humidified incubator. The dispersion of DOX-loaded PBHMONs was prepared in RPMI-1640 medium. The DOX concentration in the PBHMONs nanocarriers was 10 μg/mL. Cells were washed twice with PBS and incubated with the above DOX-loaded
Scheme 2. Synthetic scheme for PBHMONs. 3
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Fig. 1. SEM (a, b) and TEM (c, d) images of PBHMONs based on a Si source molar ratio of 50:50.
clearly show that the PBHMONs were broken down in the acidic environment and exhibited significant biodegradation after 5 d, which was faster than previous reported redox responsive mesoporous organosilica nanoparticles containing disulfide (S-S) bridges (7 d). [30,40] The SEM image (Fig. 4f) of PBHMONs after treatment with acid for 5 d also confirms the degradation of the NPs. For comparison, PBHMONs (0.1 mg/mL) were also stirred at 37 °C in PBS (pH = 7.4, up to 5 d), and the TEM images after 5 d are shown in Fig. S4. In addition, small fragments were present in the supernatant due to the degradation of the PBHMONs exposed to the acidic environment. The supernatant was further analyzed by dynamic light scattering (DLS) after centrifugation (Fig. 4g). The results supported the expectation that the as prepared PBHMONs would biodegrade faster in acidic tumor tissues, leading to rapid excretion and contributing significantly to the potential clinical translation of the system. To study the cargo loading performance of the PBHMON nanocarriers, Doxorubicin (DOX) was used as a model drug. The concentration of DOX was determined by UV–vis spectroscopy and the loading efficiency and capacity were calculated to be 86.4% and 162.9 ug-DOX∙mg/nanocarriers, respectively. To our knowledge, the drug loading efficiency of PBHMONs was much higher than those of traditional drug delivery systems (MSNs and organic micelles) [39]. In addition, the collected DOX-loaded PBHMONs was washed with PBS and almost no DOX could be detected in the washing water, indicating that most of the drug was loaded in the hollows, mesopores and inner surface of shells, instead of attached on the outer surface of shells. The biodegradation behavior and structure evolution enhanced the release properties of the PBHMONs nanoparticles. DOX-loaded PBHMONs were prepared and their release behavior was investigated using PBS solutions with different pH (pH 6.0 and 7.4, simulating tumor and normal cells) at 37 °C. As shown in Fig. 5, ∼80% of the DOX was released from the PBHMONs at pH 6.0 while only ∼20% of the DOX was released in a neutral environment after 150 h. The π-π interaction
PBMONs core/shell structure with a silica core was synthesized via the Stöber method, and PBHMONs were obtained after an etching process (Scheme 2). The molar ratio of TEOS and pH-responsive silsesquioxane affected the morphology of the bulk organosilica nanoparticles. Three different ratios (70:30, 50:50, and 30:70, TEOS:pH-responsive silsesquioxane, based on the Si source) were used to prepare PBHMONs, and the structures were analyzed by TEM. As shown in Fig. S2, the shells of the PBHMONs based on the 70:30 mol ratio were too thick, and the shells of PBHMONs based on the 30:70 mol ratio were broken after the etching process. The morphology of PBHMONs based on a molar ratio of 50:50 can be observed in Fig. 1, both the SEM and TEM images show the homogenous spherical morphology of the PBHMONs evidencing a good degree of particles size monodispersity (∼100 nm), and the hollow structure can be clearly observed from the TEM images. Dynamic light scattering (DLS) measurements were used to evaluate the particle size, and the results (Fig. S3) indicated that the diameter of the PBHMONs was ∼100 nm, which was in accordance with the findings from the microscopy images. The specific area and pore size were measured by N2 adsorption analysis. The results shown in Fig. 2 shows that the PBHMONs had a surface area of 862.5 m2/g with an average pore size of 2.2 nm. The large surface area is beneficial for drug loading and the mesopores provide a controlled diffusion path for drug payloads. The presence of organic bridges, which affect the nature of the overall structure and control the pH-responsive biodegradation of PBHMONs, was determined by energy dispersive spectrometry (EDS), and the spectrum is shown in Fig. 3. The presence of nitrogen (N) strongly supports the presence of pH-responsive organic groups in the shell of the as prepared particles. To study the structure breakdown of PBHMONs, PBHMONs (0.1 mg/mL) were stirred in acidic PBS at 37 °C (pH = 6, up to 5 days), and aliquots of the suspension were collected at regular intervals, and analyzed by TEM observation (Fig. 4). TEM images of the suspension 4
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Fig. 2. (a) N2 adsorption-desorption isotherms and (b) the corresponding pore-size distributions for PBHMONs.
Fig. 3. EDS analysis of the PBHMONs nanoparticles.
Fig. 4. Characterization of the biodegradation of PBHMONs. (a–e) TEM analysis of a suspension of PBHMONs (0.1 mg/mL, PBS, pH = 6, 37 °C) after 0, 0.5, 1, 3, and 5 d, (f) SEM images of a suspension of PBHMONs (0.1 mg/mL, PBS, pH = 6, 37 °C) after 5 d, (g) DLS analysis of the supernatant of the centrifuged dispersion of PBHMONs (0.1 mg/mL, PBS, pH = 6, 37 °C) after 1, 3, and 5 d.
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mainly distributed in cytoplasm at 2 h and nucleus at 1d, confirming the DOX leakage form the nanocarriers. The cytotoxicity of free PBHMONs was studied by MTT assay. As shown in Fig. 7a, KB cells maintained a high cell viability (> 80%) after being incubated with PBHMONs for 48 h, and the cytotoxicity of the NPs remained low when the concentration was increased. In addition, the fragments of PBHMONs that formed after acid treatment for 5 days were collected, and the cytotoxicity also remained low. These findings clearly demonstrate that both the free and degraded PBHMONs were biocompatible and nontoxic, which is important for their further biomedical application. The biodegradation of the PBHMONs and the efficient leakage of the loaded drug molecules enhanced the cytotoxicity of the DOX-loaded PBHMONs in vitro. KB cells were incubated with DOX-loaded HMS, DOX-loaded PBHMONs, and free DOX (5, 10, 20 and 40 ug-DOX/mL) for 24 h and the cell viability evaluated using MTT is shown in Fig. 7b. Compared with the non-biodegradable HMS particles loaded with DOX, the pH-responsive biodegradation of PBHMONs caused by the acid endo/lysosomes led to more efficient DOX release and subsequent diffusion of DOX from the cytoplasm to the nucleus, and it is well-known that the ultimate target of DOX is DNA to form DNA-DOX adduct to inhibit transcription and induce the death of tumor cells, which resulted in a clear difference in cytotoxicity between the DOX@HMS nanocarriers and DOX-loaded PBHMONs nanocarriers. In addition, KB cells were also incubated with pure PBHMONs for contrast, obviously high cell viability indicated that apoptosis or necrosis was not triggered by the nanoparticles in the cancer cells. Finally, TUNEL assay was carried out to demonstrate whether the above mentioned pH-responsive biodegradation of PBHMONs may result in enhancement of cell apoptosis. As shown Fig. S7, cells incubated with DOX-loaded PBHMONs have a higher apoptosis rate, which was consistent with the MTT results. To study the antitumor efficiency of DOX-loaded PBHMONs, athymic nude mice bearing KB cells were randomized into 3 treatment groups and intratumorally (i.t.) injected with ca. 50 μL of saline, PBHMONs, or DOX-loaded PBHMONs (10 mg /mL). The tumor volumes and body weights were measured over the subsequent 16 d. As shown in Fig. 8a and Fig. 8b, the tumor volume of the PBHMONs group exhibited almost no difference to the saline group, confirming that PBHMONs showed no efficient inhibition of tumor growth. In contrast, effective elimination of tumors was achieved in the group of mice treated with DOX-loaded PBHMONs owing to the effective drug release following pH responsive biodegradation. The negligible variation in the average weights of the different groups (Fig. S8) indicated that the biodegradation of PBHMONs did not affect the health of the mice. The mice were euthanized at day 16 and the major organs were
Fig. 5. DOX release profiles of DOX-loaded PBHMONs at pH = 6.0 and 7.4, and DOX-loaded HMS at pH = 6. Error bars indicate mean ± SD, n = 3.
between DOX and the pH-responsive bridges is expected to delay the drug release in normal organs/tissues, while the biodegradation of the NPs in tumor tissue facilitates the drug release; leading to relatively high biosafety, which is important for further clinical translation and application in cancer treatment. Moreover, HMS with a diameter of 140 nm (Fig. S5) were synthesized according to our previous work and loaded with DOX [41]. And the release behavior DOX-loaded HMS at pH 6.0 was also recorded, the lower release efficiency of DOX-loaded HMS demonstrated that the degradation of PBHMONs could accelerate the release of loaded DOX molecules. Owing to the red fluorescence of DOX, the intracellular activity of the DOX-loaded PBHMONs could be monitored without any additional modification. KB cells were first incubated with DOX-loaded PBHMONs in which the DOX concentration was calculated to be 10 μg mL−1. Following the removal of non-internalized NPs, cells were stained with DAPI, and then the red fluorescence of the DOX inside the cells was monitored using an inverted fluorescence microscope over periods of 2 h and 1 d. As shown in Fig. 6, the KB cells were able to take up the drug nanocarriers via an endocytosis pathway, and it was observed that DOX distributed throughout the whole cell after 2 h, while it was mainly found in the nucleus after 1 d. This observation was primarily the result of DOX leakage. To further demonstrate the distribution of DOX, co-localization fluorescence measurements was carried out using magnified cell images. As shown in Fig. S6, the fluorescence of DOX was
Fig. 6. Fluorescence images of KB cells incubated with DOX-loaded PBHMONs for 2 h (top) and 1 d (bottom). 6
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Fig. 7. Viability of KB cells after incubation with (a) PBHMONs for 48 h, and (b) DOX-loaded HMS, DOX-loaded PBHMONs, free DOX and PBHMONs for 24 h. Error bars indicate mean ± SD, n = 3. **P < 0.01, ***P < 0.001. Fig. 8. In vivo treatment with DOX-loaded PBHMONs (a) Tumor growth curves of mice after various treatments. (b) Image of tumor tissues dissected after treatment. (c) Survival curves of mice after various treatments. (d) H& E stained major organs collected from different treatment groups. Error bars indicate mean ± SD, n = 5. ***P < 0.001.
that could be stimulated by weakly acidic environments were designed and fabricated. The hollow structure of the prepared PBHMONs gave the nanocarriers a high drug loading capacity, while the use of DOX as a model drug, and the π-π interactions between the introduced pH-responsive groups in the shell and the DOX molecules, prevented the release of the drug in normal tissues. Once the DOX-loaded PBHMONs entered the tumor cells via EPR, the weakly acidic microenvironment induced the biodegradation of the PBHMONs, leading to partial drug release upon degradation of the carrier and efficient cancer cell killing, as well as rapid excretion. This study paves the way for the development of the next generation of hybrid nanocarriers tailored for cargo delivery into tumor cells for disease-targeted treatments
collected then H&E stained. As shown in Fig.8d, there was no apparent difference between the saline group, and the PBHMONs and DOXloaded PBHMONs groups, in the heart, liver, spleen, lung, and kidney, confirming that the synthesized PBHMONs caused no significant damage to the major organs. In addition, the survival rates of three further groups of athymic nude mice bearing KB cells treated using the same strategy were recorded. A lower survival rate was observed in the control groups owing to the fast growth of the tumors (Fig. 8c), demonstrating the drug delivery efficiency of PBHMONs.
4. Conclusions In summary, novel hollow tumor microenvironment (TME)-responsive biodegradable mesoporous organosilica nanoparticles (MONs) 7
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Declaration of Competing Interest
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We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled “Novel pH-responsive Biodegradable Organosilica Nanoparticles as Drug Delivery System for Cancer Therapy”. Acknowledgements We gratefully acknowledge the financial support provided by National Natural Science Foundation of China (51802127), Natural Science Foundation of Jiangsu Province (BK20170239), the Priority Academic Program Development of Jiangsu Higher Education Institutions and Jiangsu Normal University scientific research fund (16XLR005). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfa.2019.124133. References [1] W.S. Lin, W.Y. Huang, X.D. Zhou, Y.F. Ma, Toxicol. Appl. Pharmacol. 217 (2006) 252. [2] R.K. Kankala, C.G. Liu, A.Z. Chen, S.B. Wang, P.Y. Xu, L.K. Mende, C.L. Liu, C.H. Lee, Y.F. Hu, ACS Biomater. Sci. Eng. 3 (2017) 2431–2442. [3] R.K. Kankala, H. Zhang, C.G. Liu, K.R. Kanubaddi, C.H. Lee, S.B. Wang, W. Cui, H.A. Santos, K.L. Lin, A.Z. Chen, Adv. Funct. Mater. (2019) 1902652. [4] L. Tang, T.M. Fan, L.B. Borst, J. Cheng, ACS Nano 6 (2012) 3954. [5] A. Agostini, L. Mondragon, A. Bernardos, R. Martinez-Manez, M.D. Marcos, F. Sancenon, J. Soto, A. Costero, C. Manguan-Garcia, R. Perona, M. Moreno-Torres, R. Aparicio-Sanchis, J. Ramon Murguia, Angew. Chem. Int. Ed. 51 (2012) 10556. [6] H. Mekaru, J. Lu, F. Tamanoi, Adv. Drug Del. Rev. 95 (2015) 40. [7] C. Coll, L. Mondragon, R. Martinez-Manez, F. Sancenon, M. Dolores Marcos, J. Soto, P. Amoros, E. Perez-Paya, Angew. Chem. Int. Ed. 50 (2011) 2138. [8] C.G. Liu, Y.H. Han, J.T. Zhang, R.K. Kankala, S.B. Wang, A.Z. Chen, Chem. Eng. J. 370 (2019) 1188–1199. [9] K.C. Kao, C.Y. Mou, Microporous Mesoporous Mater. 169 (2013) 7. [10] N.Ž. Knežević, J.O. Durand, Nanoscale 7 (2015) 2199. [11] W.R. Zhao, M.D. Lang, Y.S. Li, L. Li, J.L. Shi, J. Mater. Chem. 19 (2009) 2778. [12] Y. Chen, H.R. Chen, L.M. Guo, Q.J. He, F. Chen, J. Zhou, J.W. Feng, J.L. Shi, ACS Nano 4 (2010) 529.
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