sodium alginate for anti-cancer drug delivery application

Applied Surface Science 440 (2018) 853–860

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Surface modification of graphene oxide nanosheets by protamine sulfate/sodium alginate for anti-cancer drug delivery application Meng Xie a,b, Feng Zhang c, Lijiao Liu a, Yanan Zhang a, Yeping Li a, Huaming Li d,⇑, Jimin Xie c,⇑ a

School of Pharmacy, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, PR China School of Material Science and Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, PR China c School of Chemistry and Chemical Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, PR China d Institute for Energy Research, Jiangsu University, Zhenjiang 212013, PR China b

a r t i c l e

i n f o

Article history: Received 24 August 2017 Revised 1 January 2018 Accepted 22 January 2018 Available online 31 January 2018 Keywords: GO nanosheets Surface modification LbL technique Drug delivery In vitro

a b s t r a c t In order to improve the efficiency of anticancer drug delivery, a graphene oxide (GO) based drug delivery system modificated by natural peptide protamine sulfate (PRM) and sodium alginate (SA) was established via electrostatic attraction at each step of adsorption based on layer-by-layer self-assembly. The nanocomposites were then loaded with anticancer drug doxorubicin hydrochloride (DOX) to estimate the feasibility as drug carriers. The nanocomposites loaded with DOX revealed a remarkable pHsensitive drug release property. The modification with protamine sulfate and sodium alginate could not only impart the nanocomposites an improved dispersibility and stability under physiological pH, but also suppress the protein adhesion. Due to the high water dispersibility and the small particle size, GO-PRM/SA nanocomposites were able to be uptaken by MCF-7 cells. It was found that GO-PRM/SA nanocomposites exhibited no obvious cytotoxicity towards MCF-7 cells, while GO-PRM/SA-DOX exhibited better cytotoxicity than GO-DOX. Therefore, the GO-PRM/SA nanocomposites were feasible as drug delivery vehicles. Ó 2018 Elsevier B.V. All rights reserved.

1. Introduction Cancer has been recognized as one of the major malignant diseases concerns worldwide. The conventional cancer treatment is mainly surgery, chemotherapy, and radiotherapy. Although chemotherapy has been widely used in clinical treatment, most chemotherapeutic drugs are lowly specific and highly toxic, leading to side effects and systemic toxicity [1,2]. In order to decrease the adverse effects and to improve the therapeutic efficiency of chemotherapeutic drugs, numerous drug delivery systems have been developed, such as liposomes [3,4], polymeric micelles [5– 9], silica nanoparticles [10–14], carbon based materials [15–17], metal–organic frameworks [18–20], and so on. The nanomaterial-mediated drug delivery systems have generated great interest because of their notable superiority in enhancing antitumor efficiency and decreasing systemic toxicity in cancer therapy [1,2]. Among the various nanomaterials of different sizes and shapes, graphene oxide (GO) has attracted considerable attention in drug delivery owing to its superior ⇑ Corresponding authors. E-mail addresses: [email protected] (H. Li), [email protected] (J. Xie). https://doi.org/10.1016/j.apsusc.2018.01.175 0169-4332/Ó 2018 Elsevier B.V. All rights reserved.

biocompatibility and high surface area practicable for drug loading [21–23]. In recent years, an amount of studies have been reported for modificated GO as drug carrier. For example, Nasrollahi et al. synthesized Transferrin/Poly (allylamine hydrochloride)-functionalized graphene oxide for targeted delivery of docetaxel [24]. Wang et al. Prepared thermo-sensitive graphene oxide–polymer nanoparticle hybrids as a carrier for drug delivery [25]. Cao et al. developed folic acid-conjugated GO as a transporter of chemotherapeutic drug and siRNA for reversal of cancer drug resistance [26]. These developments suggest that it is possible to construct GO based drug delivery system for drug delivery. Despite of various advantages, GO is subjected to aggregation in physiological conditions, which severely limited its application in drug loading and biomedical application. Further functionalization of GO is therefore needed [27,28]. The method of layer-by-layer (LbL) technique has emerged as a novel approach for surface modification [29,30]. The technique involves alternative immobilization of polymers with opposite charges on the surface of the bases via electrostatic attractions to form functional multilayers. For example, Ramasamy et al. developed lipid-polymer hybrid nanoparticles via LbL coating for drug

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delivery [31]. Haidar et al. prepared alginate and chitosan modified liposomes for delivery of biomacromolecules [32]. In addition, polyethylenimine /poly(acrylic acid) coated rGO sheets were also prepared as reactive precursors [33]. We previously prepared nanocomposites of graphene oxide with chitosan/ dextran or chitosan/sodium alginate as drug carriers and the functionalization significantly improved the stability of the drug loaded system [34,35]. Although these literatures have been reported before, seldom attention was paid on the LbL immobilization of natural peptides or proteins on nano-sized GO surfaces for drug delivery applications. In this work, a natural peptide protamine sulfate (PRM) was selected and to prepare multi-layered nanocomposites with oppositely charged sodium alginate (SA) by layer-by-layer deposition on GO nanosheets (Scheme 1). Protamine sulfate is a biodegradable cationic peptide derived from fish sperm and it is positively charged owing to their high arginine content [36,37]. It occurs naturally in sperm and has been sometimes applied for surface coating [38]. More importantly, protamine contains arginine-rich nuclear localization signal (NLS), which can specifically direct the particles to the nucleus, thus has an application in gene delivery [39,40]. Sodium alginate (SA), a linear anionic natural polysaccharide, which is biocompatible, and biodegradable under normal physiological conditions, has been widely used in biomedical applications [41,42]. GO surfaces were negatively charged, which was available for the immobilization of cationic protamine sulfate and anionic sodium alginate via electrostatic interactions. The modification was demonstrated via AFM and zeta potential measurements. The resultant GO-PRM/SA nanocomposites were applied to deliver doxorubicin (DOX), an effective anticancer drug in physiological environments. The drug release behaviors at different pH values have been evaluated. Finally, the cellular uptake of fluorescent nanocomposites and the cytotoxicity of the DOX loaded ones were investigated by CLSM and MTT assay respectively to evaluate their potential for anti-cancer drug delivery.

reported in our works [34,35]. Nano-sized GO sheets were obtained via sonication using an ultra-sonic cell disrupter system for 1 h. Protamine modified GO was prepared with 1:4 weight ratio of GO and protamine. Typically, protamine sulfate was dissolved deionized water (1 mg/mL). Then, GO suspension (0.2 mg/mL) was slowly added to the solution with stirring for 20 min. After that, the suspension was filtered via a membrane (0.22 lm). The obtained solid was redistributed in purified water and filtered three times to remove the residue protamine, and then dispersed in pure water to obtain protamine functionalized GO suspension. The GO-PRM/SA complex was synthesized via the same procedure. The weight ratio of protamine sulfate and sodium alginate was at 1:1.

2.3. Characterization Fourier transform infrared (FT-IR) spectra were registered with a Nicolet Model Nexus spectrometer to identify the functional groups of the composites. The zeta potential analysis was performed on the Malvern ZEN3600 equipment. AFM images were carried out by a Multimode 8 atomic force microscopy system (Bruker, USA) to investigate the morphology of the samples.

2.4. Preparation of fluorescent nanocomposites Fluorescein isothiocyanate (FITC) was conjugated to protamine sulfate via covalent bond to prepare protamine-FITC for imaging. Firstly, 100 mg protamine sulfate was dissolved in 10 mL carbonate buffer (pH 9.0). Then, 0.1 mL FITC solution (in ethanol, 10 mg mL 1) was added to the solution and was stirred for 12 h in the dark. After that, unreacted FITC was removed by dialysis in purified water in the dark for 24 h. Then, protamine-FITC and sodium alginate were deposited on the surface of GO to form GO-PRM-SA fluorescent nanocomposites by the same procedure as described above.

2. Experimental section 2.1. Materials

2.5. Non-specific protein adsorption

Protamine sulfate salt was purchased from Sigma-Aldrich (Steinheim, Germany). Sodium alginate was purchased from Sinopharm Chemical Reagent Company (Shanghai, China). Doxorubicin hydrochloride (DOX) was purchased from HVSF United Chemical materials Co. Ltd. (Beijing, China).

By using Bovine Serum Albumin (BSA) as a model protein, we investigated the protein attachment on GO-PRM/SA nanocomposites. Generally, BSA was dispersed with PBS buffer (pH 7.4) to a concentration of 1 mg mL 1. 2 mL of the formed BSA solution was mixed with 5 mL GO or GO-PRM/SA suspension (at a GO concentration of 0.2 mg mL 1) and stirred for 24 h. Following this, the unbounded BSA molecules in the mixture was separated with the aid of centrifugation. Finally, the measurement of the content of protein absorbed was obtained via UV–vis spectroscopy at 280 nm based on the BSA in feed subtracting the unbounded BSA molecules.

2.2. Production of GO-PRM/SA The GO-PRM/SA composites were fabricated by LbL technology. The preparation of GO nanosheets has been previously

2.6. Drug loading studies

Scheme 1. Illustration showing the synthesis of GO-PRM/SA and DOX loading.

To prepare the drug loaded nanocomposites, 2 mL PBS solution of DOX (0.5 mg mL 1, pH 7.4) was directly added to the suspensions of GO, GO-PRM and GO-PRM/SA (2.5 mL, at a GO concentration of 0.2 mg mL 1) respectively, and stirred for 24 h. The unbound DOX in the supernatant was removed by extensive centrifugation (13,000 rpm, 30 min). The amount of DOX in the supernatant was determined with a UV spectrophotometer at the wavelength of 480 nm. The extent of DOX loading could be evaluated by subtracting the amount of drug in the supernatant from the original DOX solution.

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2.7. Drug release behavior The release behavior of DOX was investigated at two different pH values (7.4 and 5.0) through dialysis. Briefly, DOX-loaded nanocomposites in PBS buffer (0.2 mg mL 1, 1 mL) were placed in dialysis bags, and the bags were then saturated into 20 mL of release medium in 50 mL centrifuge tubes. The samples were incubated in an incubator shaker at 37 °C. At definite time points, the whole medium was withdrawn and equal volume of fresh buffer was replenished. The fluorescence of DOX was determined to calculate the amounts of released DOX. 2.8. Cytotoxicity measurement The in vitro cytotoxicity investigation was performed using MTT assays. Generally, the MCF-7 cells were plated in a 96-well plate and incubated overnight to allow cell attachment. Afterwards, different concentrations of blank nanocomposites or DOX loaded nanocomposites were added to each well and incubated for 48 h. Following this, the supernatant was removed and the cells were treated with 20 lL of MTT solution (5 mg/mL). After another 4 h, the medium was aspirated off and DMSO (200 lL well 1) was added. The measurement of the suspension optical density (OD) was performed on an automated microplate reader 550 (Bio-Rad, USA). 2.9. Cellular uptake study The in vitro cellular uptake study was performed using CLSM images. Generally, MCF-7 cells were plated in glass-bottom culture dishes and incubated overnight to allow cell attachment. Afterwards, FITC-labeled GO-PRM/SA nanocomposites were added to each dish and incubated for 4 h at 37 °C. For uptake inhibition evaluation, the cells were pre-incubated at 4 °C for 1 h, and then incubated with FITC-labeled GO-PRM/SA nanocomposites for 4 h at 4 °C. After this step, the cells were washed for three times, fixed with paraformaldehyde and stained by DAPI. The images were obtained by CLSM. 3. Results and discussion 3.1. Characterization of GO-PRM/SA 3.1.1. FT-IR analysis Fourier-transforms infrared (FTIR) spectrum peaks were recorded to examine the formation of nanocomposites. As

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illustrated in Fig. 1A, the absorption band of GO at 1732, 1624, 1074 and 1412 cm 1 are ascribed to stretching frequency of C@O (carboxylic groups), AC@CA, CAO and CAOH, respectively [43]. In the spectrum of protamine sulfate, the bands for amide I and amide II bands were observed at 1655 and 1541 cm 1 respectively [44]. The peak at 1088 cm 1 was attributed to the CAN adsorption [45]. After the generation of GO-PRM, the peaks at 1655, 1535 and 1088 cm 1 altered to 1662, 1539 and 1079 cm 1 respectively. The altered absorbance supported protamine sulfate conjugation to GO. In the spectrum of sodium alginate (Fig. 1B), the absorbance peak at 1610 cm 1 and 1419 cm 1 comes from the carboxylate (COO) vibration. The band at 1128 cm 1 originates from CAO vibration [46]. Similarly, after the generation of GO-PRM/SA, the peaks of sodium alginate at 1419 and 1128 cm 1 respectively transferred to 1416 and 1078 cm 1, which is attributed to the conjugation of sodium alginate with GO-PRM nanocomposites.

3.1.2. Particle size analysis The particle sizes and thickness of the nanocomposites were characterized by dynamic light scattering (DLS) and AFM. According to DLS analysis, the mean size of GO sheets was 247.62 ± 5.62 nm, indicating GO were nano-sized sheets. After the layer-by-layer deposition of protamine sulfate and sodium alginate, the particle size increased significantly to 278.67 ± 4.86 nm and 361.89 ± 7.66 nm, respectively, suggesting the successful deposition of the polyelectrolytes. Fig. 2 shows the representative images of GO, GO-PRM and GOPRM/SA by AFM. As shown, the thickness of 1 nm was observed for GO sheets. Meanwhile, The sheets were nanosized after the sonication. After the modification of protamine sulfate, the thickness became greater, which was about 20 nm. The increase in thickness was due to the coating of protamine sulfate layer on the surface of GO. When sodium alginate was further deposited, the thickness altered dramatically from 20 nm to 40 nm, indicating sodium alginate has successfully deposited. In our previous work [35], chitosan and sodium alginate were chosen as polyelectrolytes to prepare GO based nanocomposites via LbL modification. After the modification of chitosan, the thickness of GO sheets only increased to 6 nm, and the deposition is less homogeneous than the deposition of protamine sulfate (Fig. 2B), indicating strong interaction between protamine sulfate and GO sheets.

Fig. 1. (A) FTIR spectra of GO, protamine sulfate and GO-PRM. (B) FTIR spectra of GO-PRM, sodium alginate and GO-PRM/SA.

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Fig. 2. AFM images of GO (A), GO-PRM (B) and GO-PRM/SA (C).

3.1.3. Determination of the surface charge The formation of LbL multilayers on GO nanosheets was investigated by zeta potential measurements. The initial zeta potential of GO was 33.25 mV. When protamine was deposited, the potential varies from negative to positive (30.13 mV), which resulted from the polycationic charge of protamine. When sodium alginate was further deposited, the zeta potential reversed to negative ( 24.47 mV) again. The results indicated that protamine sulfate and sodium alginate were successfully coated. 3.2. Stability studies Fig. 3 showed the stability characterization of different samples in different solutions after 48 h. As depicted, pure GO without modification showed a fast aggregation in PBS and cell culture medium after 48 h. After the modification of protamine sulfate, the dispersion of GO-PRM had a little improvement in cell culture medium, but it also aggregated in PBS solution. This indicated that protamine sulfate modification alone was not a good method to improve the stability of GO nanosheets. Nevertheless, when GOPRM was further functionalized with sodium alginate, the GOPRM/SA nanocomposites displayed a homogenous dispersion in

the three kinds of solutions. From these results, we can infer that PRM/SA modification could enhance the stability of GO nanosheets under physiological pH. 3.3. Non-specific protein adsorption As is acknowledged, non-specific protein adsorption could trigger the internalization of nanocomposites by macrophages and generate detrimental inflammatory reaction. Thereby, it is indispensable to construct new drug delivery carriers that exhibit low degrees of non-specific protein adhesion. Bovine serum albumin (BSA, albumin bovine V, sigma) was employed as a model to detect the non-specific protein binding on GO nanosheets and GO-PRM/ SA nanocomposites. The percentage of protein adsorption for GO was 140.64% (w/w) after 24 h. However, the modification with protamine sulfate and sodium alginate could suppress the nonspecific protein adsorption, with the adsorption amount of 45.6% and 7.61% respectively. It is known that materials possessing hydrophilic surface usually show low non-specific protein adsorption, while hydrophobic surface show high non-specific protein adsorption [47]. The results indicate that surface functionalization of GO by protamine sulfate and sodium alginate immobilization

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Fig. 3. The stability of GO, GO-PRM and GO-PRM/SA in purified water (left), PBS (middle) and PRMI-1640 cell culture with 10% serum (right), respectively.

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Wavelength (nm) Fig. 5. Fluorescence spectra of DOX (a), GO-DOX (b), GO-PRM-DOX (c) and GOPRM/SA-DOX (d). Fig. 4. The drug loading amount of GO, GO-PRM and GO-PRM/SA. The insert show the stability images of the three samples (from left to right) after DOX loading.

could suppress the non-specific protein adsorption due to the improvement of hydrophilicity of GO surface. 3.4. Drug loading DOX loaded nanocomposites were prepared by simple mixing method. The content of DOX loading was evaluated using UV–Vis spectra. As shown in Fig. 4, the DOX loading percentage was determined to be as high as 155.81%. However, after drug loading, GO nanosheets generated serious precipitation (Fig. 4 insert). For comparison, the DOX loading amount (W/W) for GO-PRM and GOPRM/SA were evaluated with 83.54% and 129.78% respectively. After the decoration of PRM/SA, the solubility of the nanocomposites had a remarkable enhancement, and no obvious precipitation was seen on DOX-GO-PRM/SA. Taken together, all the results presented the functionalization of PRM/SA effectively facilitated the stability of GO nanosheets loaded by DOX. As was reported, GO had a fluorescence quenching effect on DOX molecules. Photoluminescence spectra of the samples were

presented in Fig. 5. The suspension of the samples were of equal DOX concentration. For free DOX, a maximum fluorescence emission at 580 nm was seen (kex = 488 nm). In contrast, the quenching property of GO to the fluorescence of DOX was remarkable. This indicated that DOX has successfully adsorbed onto GO nanosheets. When DOX was loaded into GO-PRM/SA, the characteristic was similar in fluorescence quenching. The analysis demonstrated that DOX was attached on GO nanosheets, not immobilized on protamine sulfate or sodium alginate surface. 3.5. pH sensitive drug release behavior The drug release profiles of the DOX-loaded noanocomposites were studied at pH 5.0 as well as at pH 7.4 (Fig. 6), which represents the acidic tumor microenvironment and normal physiological environment, respectively. At the physiological pH of 7.4, 28.1% and 26.5% of DOX released from DOX-loaded GO nanosheets and DOX-loaded noanocomposites respectively after 168 h. By comparison, at pH 5.0, 55.6% and 49.5% drug release from DOX-loaded GO nanosheets and DOX-loaded noanocompos-

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Fig. 6. The release profiles of DOX from GO and GO-PRM/SA in PBS buffer (pH 7.4, pH 5.0) at 37 °C.

Fig. 8. In vitro cytotoxicity of DOX and DOX-loaded nanocomposites. MCF-7 cells were incubated with the nanocomposites for 48 h.

ites have been achieved respectively. The faster DOX release at lower pH conditions may be resulted from the protonation of DOX molecules and reduced force between DOX and GO nanosheets. Furthermore, compared to DOX-GO, a little faster DOX release speed was observed in DOX-GO-PRM/SA, especially before 96 h, which may be ascribed to the increased diffusion of DOX from the nanocomposites. Meanwhile, the GO-DOX was unstable in physiological environment, thus it was not a good choice for application. Conversely, the GO-PRM/SA system has a perfect stability, so it is a promising material to suit the application. The DOX release profile from GO-PRM/SA-DOX is similar to our previous reported GO-CS/SA-DOX system [35], indicating that the deposition of polyelectrolytes did not obviously affect the DOX release rates. As the tumor tissues have an acidic environment, the pH sensitive drug release property could facilitate the drug release in the tumor cells, which may be efficient for the treatment of cancer.

MCF-7 cells were treated with free DOX or equivalent DOX concentration of DOX-loaded nanocomposites for 48 h. As depicted in Fig. 7, after 48 h incubation, neither pure GO or GO-PRM/SA was toxic to MCF-7 cells at various concentrations. In addition, the cytotoxicity of DOX-loaded samples were further investigated within the tested concentration ranges. As shown in Fig. 8, all the three samples limited cell viability with increasing drug doses. As both pure GO and GO-PRM/SA did not induce serious cytotoxicity in MCF-7 cells, the observed cytotoxicity was caused by the released DOX molecules from DOX loaded samples. Though the free DOX inhibited the cancer cells much better, it may also inhibit the normal ones. The DOX loaded GO-PRM/SA was more cytotoxic than GO-DOX. For DOX loaded GO-PRM/SA, the improved cytotoxicity compared with GO-DOX may be caused by the fast drug release from GO-PRM-SA-DOX at low pH values. 3.7. Cellular fluorescence imaging

3.6. Cellular cytotoxicity assay MTT cell viability assay was employed to detect the cytotoxicity of pure nanocomposites and DOX-loaded nanocomposites.

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The cellular uptake behavior of GO-PRM/SA nanocomposites were further explored. Green fluorescent dye FITC molecules were conjugated to protamine sulfate by covalent bond to prepare fluorescent nanocomposites. The nuclei were stained blue with DAPI. As is reported, protamine presents unique nuclear localization signals (NLS), thus is able to entry into the nucleus [39,40]. This is confirmed by CLSM (Fig. 9a), where strong green fluorescence could be seen from the nuclei of MCF-7 cells after the treatment of protamine-FITC. In comparison, strong green fluorescence signals from FITC labeled GO-PRM-SA nanocomposites could also be seen, but mostly distributed in the cytoplasm (Fig. 9b), further indicating successful deposition of protamine on the surface of GO. We then evaluated the uptake mechanism of GO-PRM-SA nanocomposites. Cells were treated with FITC labeled GOPRM-SA nanocomposites at 4 °C to inhibit energy dependent cellular uptake. As shown in Fig. 9c, the uptake of GO-PRMSA at low temperature was almost suppressed. It was confirmed that GO-PRM/SA composites were taken up via energy-dependent endocytosis, which would be due to the small particle size (<500 nm) and high water dispersibility of the nanocomposites. Based on high water dispersibility, good biocompatibility and effective internalization by MCF-7 cells, it was implied that GO-PRM/SA should be ideal candidates for biomedical applications.

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Fig. 9. Fluorescent images of PRM-FITC (a) and GO-PRM/SA-FITC (b, c) intracellular accumulation. MCF-7 cells were incubated with GO-PRM/SA-FITC for 4 h at 37 °C (b) and 4 °C (c), respectively.

4. Conclusion In summary, the present study investigated a novel drug delivery system comprising graphene oxide (GO), protamine sulfate (PRM) and sodium alginate (SA). We have studied the synthesis, characterization, and in vitro toxicity of GO-PRM/SA nanocomposites. The GO-PRM/SA nanocomposites possess high dispersibility, stability and suppressed non-specific protein adsorption in physiological environments. Analysis of drug loading and drug release assay demonstrated that the system had a high loading capacity and quick release behavior at low pH values. In vitro cytotoxicity results turned out that GO-PRM/SA nanocomposites exhibited no obvious cytotoxicity towards MCF-7 cells. Due to the high water dispersibility and the small particle size, GO-PRM/SA nanocomposites were able to be taken up by MCF-7 cells, and GO-PRM/SA-DOX exhibited better cytotoxicity than GO-DOX. Hence, the study may provide new prospects for utilizing the GO-based nanocomposites coated by natural peptides or proteins in biomedicine field. Acknowledgements This work is financially supported by the National Natural Science Foundation of China (No. 21506079, 21676129), Nature Science Foundation of Jiangsu Province – China (No.

BK20140577), Jiangsu University Scientific Research Funding – China (14JDG164) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References [1] R.K. Jain, T. Stylianopoulos, Delivering nanomedicine to solid tumors, Nat. Rev. Clin. Oncol. 7 (2010) 653–664. [2] J.A. Barreto, W. O’Malley, M. Kubeil, B. Graham, H. Stephan, L. Spiccia, Nanomaterials: applications in cancer imaging and therapy, Adv Mater. 23 (2011) 18–40. [3] Y.S. Feng, C.Y. Sun, Y.Y. Yuan, Y. Zhu, J.Y. Wan, C.K. Firempong, E. Omari-Siaw, Y. Xu, Z.Q. Pu, J.N. Yu, X.M. Xu, Enhanced oral bioavailability and in vivo antioxidant activity of chlorogenic acid via liposomal formulation, Int. J. Pharm. 501 (2016) 342–349. [4] Y.W. Wang, S.C. Wang, C.K. Firempong, H.Y. Zhang, M.M. Wang, Y. Zhang, Y. Zhu, J.N. Yu, X.M. Xu, Enhanced solubility and bioavailability of Naringenin via liposomal nanoformulation: preparation and in vitro and in vivo evaluations, AAPS Pharmscitech 18 (2017) 586–594. [5] S. Biswas, P. Kumari, P.M. Lakhani, B. Ghosh, Recent advances in polymeric micelles for anti-cancer drug delivery, Eur. J. Pharm. Sci. 83 (2016) 184–202. [6] J.M. Pan, R.R. Wu, X.H. Dai, Y.J. Yin, G.Q. Pan, M.J. Meng, W.D. Shi, Y.S. Yan, A hierarchical porous bowl-like PLA@MSNs-COOH composite for pH-dominated long-term controlled release of doxorubicin and integrated nanoparticle for potential second treatment, Biomacromolecules 16 (2015) 1131–1145. [7] H.Y. Zhang, W.Q. Xu, Y.W. Wang, E. Omari-Siaw, Y. Wang, Y.Y. Zheng, X. Cao, S. S. Tong, J.N. Yu, X.M. Xu, Tumor targeted delivery of octreotide-periplogenin conjugate: synthesis, in vitro and in vivo evaluation, Int. J. Pharm. 502 (2016) 98–106.

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