âBottom-upâ Construction of Multi-Polyprodrug-Arm Hyperbranched

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“Bottom-up” construction of multi-polyprodrugarm hyperbranched amphiphiles for cancer therapy Pei Sun, Dong Chen, Hongping Deng, Nan Wang, Ping Huang, Xin Jin, and Xinyuan Zhu Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 25 Apr 2017 Downloaded from http://pubs.acs.org on May 1, 2017

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Bioconjugate Chemistry

“Bottom-up” Construction of Multi-polyprodrug-arm Hyperbranched Amphiphiles for Cancer Therapy Pei Sun, Dong Chen, Hongping Deng, Nan Wang, Ping Huang, Xin Jin*, Xinyuan Zhu School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China.

* Corresponding authors. E-mail: [email protected]. Telephone: +86-21-34203400. Fax: +86-21-54741297.

ACS Paragon Plus Environment

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: Despite of great advantages of polymer-drug conjugates (PDC) in cancer therapy, the controlling of drug loading site and degree via a facile approach remains a great challenge.

Herein, combination the controllability of the “bottom-up” strategy and the

stability of multi-arm hyperbranched amphiphiles, we have developed a novel multipolyprodrug-arm hyperbranched amphiphiles (H40-star-(PHCPTMA-b-PMPC), hPCM) via reversible addition-fragmentation chain transfer (RAFT) polymerization for cancer therapy. The hPCM was constructed via two-step polymerization of an acid-labile prodrug monomer and a zwitterionic monomer, respectively. By using a H40 macroRAFT agent, 10hydroxycamptothecine (HCPT) prodrug monomers were directly polymerized via the “bottom-up” strategy as polyprodrug-arms inner-shell of hPCM with homogeneous drug distribution. The drug loading content can be facile tuned through varying the feed ratio of HCPTMA/H40 macroRAFT agent. Finally, the poly(zwitterions) hydrophilic outer-shell of hPCM was formed by RAFT polymerization of zwitterionic monomer to ensure the preferable biocompatibility. When dissolving in dilute solution, unimolecular micelles of hPCM can be obtained, which endow a desirable stability for the micelles. The effective cellular internalization, extended blood retention time, considerable accumulation in tumor tissue, and excellent anticancer activity of the hPCM micelles have been evaluated both in vitro and in vivo. This novel multi-polyprodrug-arm hyperbranched amphiphiles constructed via the “bottom-up” strategy may open up new horizons for exploring next-generation PDC based drug delivery systems.


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INTRODUCTION Recent years have witnessed the tremendous progress in the areas of drug delivery.1-3 Up to now, as a promising drug delivery vehicle, synthetic polymeric nanocarriers have been widely applied to deliver hydrophobic anticancer drugs, aiming to achieve better water solubility, prolong blood retention time and improve the accumulation of drug in tumor sites.4-8 Considering the pharmacokinetics in vivo, chemical conjugation of small molecule drugs to polymeric carriers has exhibited much more stability and controllability than physical encapsulation.9-15 The classic approach for the synthesis of PDC involves anchoring small drugs to the synthesized polymers via “conjugate to” approach, which requires tedious synthetic procedures with limited control of the drug loading sites and capacity. A preferable approach is, therefore, the ‘‘bottom-up’’ strategy, which proceeds with high inverts the conventional “conjugate to” approach and instead directly incorporates the drugs during the synthesis of the polymer by the polymerization of polymerizable prodrug monomers. Indeed, such “bottom-up” strategy has been successfully used to construct linear PDC.16-20 For instance, Liu and co-workers synthesized linear polyprodrug amphiphiles PEG-b-PCPTM via the “bottom-up” strategy. In this case, the reduction-cleavable camptothecin prodrug monomer was directly polymerized using PEG-based macroRAFT agent via RAFT technique.21 Another prominent example is the “bottom-up” synthesis of linear PDC by the ring-opening polymerization of PEG and prodrug monomers consisting of a cyclic polymerizable group.22 In this case, more than one drug were copolymerized and the drug loading can also be readily tuned by varying the monomer/initiator feed ratio. However, the efficacy of the polymer micelles based on linear amphiphilic block polymers is often limited by their instability in vivo due to the interactions with the serum components, especially the dilution of blood.23-26 To overcome such instability associated with linear polymeric micelles, attention has been


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directed toward the synthesis of amphiphilic multi-arm hyperbranched copolymers.27-33 It has been demonstrated that amphiphilic multi-arm hyperbranched polymers can exist as unimolecular micelles composed of a hydrophobic core and hydrophilic shell in aqueous solution.34-42 Due to such covalently interconnected core-shell structure, unimolecular micelles can maintain excellent stability regardless of the extremely high dilution condition and other microenvironment changes, which is beneficial for the drug delivery efficacy. Although plenty of drug-conjugated unimolecular micelles have been investigated as stable drug delivery systems, most of them were synthesized via the conventional “conjugate to” approach.43-48 Thus, it is of great interest to construct drug-conjugated unimolecular micelles by the “bottom-up” approach for cancer therapy. Very recently, Liu and co-workers made an important contribution to this area by reporting a novel theranostic unimolecular micelle system with a hyperbranched polyprodrug core constructed via the “bottom-up” approach.49 From the view of the core-shell structure of the unimolecular micelles, the large number of arms around the core may also provide enough sites for drugs. Definitely, the “bottom up” construction and in vivo antitumor efficiency of such multi-polyprodrug-arm hyperbranched amphiphiles have not been reported yet. Herein, combination of the controllability of the “bottom-up” strategy and the stability of multi-arm hyperbranched polymers, we report a novel multi-polyprodrug-arm hyperbranched amphiphiles, hPCM, based on H40 core with an acid-labile polyprodrug-arms inner-shell and a biocompatible poly(zwitterions) outer-shell for cancer therapy (Figure 1). For this purpose, an acid-labile HCPT prodrug monomer (HCPTMA) was firstly synthesized and polymerized by RAFT technique as a polyprodrug-arms inner-shell in the presence of a H40 macroRAFT agent. By changing the feed ratio of HCPTMA/H40 macroRAFT agent, the drug loading content can be facile tuned. Then, zwitterionic monomer 2-methacryloyloxyethyl phosphorylcholine (MPC) was polymerized as a hydrophilic outer-shell to prolong blood


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retention time. The resulting multi-polyprodrug-arm hyperbranched amphiphiles hPCM can exist as covalently interconnected unimolecular micelles under the dilute solution with excellent stability. The hPCM micelles can effectively internalized by MCF-7 cells and exhibit comparable cytotoxicity compared with free HCPT. More significantly, the stability of the hPCM micelles ensures a longer retention time than free HCPT in the bloodstream, facilitating the accumulation of the micelles in tumor tissue. These advantages of the hPCM micelles result in a superior antitumor efficacy in vivo.

Figure 1. A schematic illustration of multi-polyprodrug-arm hyperbranched amphiphiles (H40-star-(PHCPTMA-b-PMPC), hPCM) from fabrication, self-assembly to delivery.

RESULTS AND DISCUSSION Synthesis

and

Characterization

of

Multi-polyprodrug-arm

Hyperbranched

Amphiphiles hPCM. As for the preparation of advanced macromolecular architectures, the ACS Paragon Plus Environment

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RAFT technique has been widely employed as an effective synthetic approach.50, 51 In the current study, multi-polyprodrug-arm hyperbranched amphiphiles hPCM was synthesized from the sequentially polymerization of HCPTMA and MPC under conventional radical initiation in the presence of H40 macroRAFT agent (Figure 2). As shown in Figure. 2A, a hydrophobic anticancer drug, 10-hydroxycamptothecin (HCPT), is used as a model drug. The polymerizable prodrug monomer HCPTMA with an acid cleavable ester bond was prepared through the reaction of phenyl hydroxyl group of HCPT with methacrylic anhydride.

Figure 2. Synthetic routes toward multi-polyprodrug-arm hyperbranched amphiphiles, H40star-(PHCPTMA-b-PMPC) (hPCM). (A) Synthesis of prodrug monomer HCPTMA. (B) Synthesis of hPCM via two-step RAFT polymerization of prodrug monomer HCPTMA and zwitterionic monomer MPC. As for the hyperbranched core, Boltorn® H40 (H40), the 4th generation hyperbrnched


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polyester with hydroxyl terminal groups, was modified to be the H40 macroRAFT agent via two-step procedures (Figure 2B). The H40-MAh with terminal vinyl groups was prepared firstly through the reaction of terminal hydroxyl groups of H40 with maleic anhydride (MAh). Then H40 macroRAFT agent was synthesized via the thiol-ene Michael addition reaction between H40-MAh and DTBA. The chemical structures of H40-MAh and H40 macroRAFT agent were characterized by using 1H NMR nuclear magnetic resonance spectroscopy as shown in Figure 3A,B.

Figure 3. 1H NMR spectra of (A) H40-MAh (in d6-DMSO), (B) H40 macroRAFT agent (in d6-DMSO), (C) hPC-3 (in d6-DMSO), and (D) hPCM (in D2O). As shown in Figure 3A, the resonance signals at δ = 6.2-6.4 ppm, ~ 13.0 ppm are corresponding to the protons of double bonds and carboxyl groups from the MAh moiety, respectively. In addition, compared to the FTIR spectrum of H40 (Figure S3A), a new peak at 1659 cm-1 appeared in spectrum of H40-MAh can be ascribed to the C=C stretching vibration. From Figure 3B, we can clearly observe the complete disappearance of the peaks at


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δ = 6.2-6.4 ppm ascribed to double bonds in H40-MAh and the appearance of new peaks at δ = 7.5-8.0 ppm characteristic of phenyl protons in DTBA, which indicates that the terminal vinyl groups of H40-MAh have reacted with DTBA. Moreover, in the FTIR spectrum of H40 macroRAFT agent (Figure S3A), compared to the spectrum of H40-MAh, the C=C stretching absorption band at 1659 cm-1 completely disappeared. These results demonstrate that H40 macroRAFT agent has been synthesized successfully. In general, there are about 60 terminal hydroxyl groups on the surface of H40. If these hydroxyl groups are completely converted into the DTBA moiety, the equivalent DTBA moiety should be observed. According to the content of sulfur element (12.4%) analyzed by element analysis, the average number of DTBA moiety per H40 macroRAFT agent is determined to be about 30, less than the number of terminal hydroxyl groups of H40. The likely reason for the incomplete conversion of hydroxyl groups is that the steric hindrance of the two adjacent hydroxyl groups is unfavorable for the reaction. After that, RAFT polymerization of prodrug monomer HCPTMA with an acid-labile ester bond was performed on the surface of H40 macroRAFT agent to prepare multi-ployprodrugarm hyperbranched core H40-star-PHCPTMA (hPC) with H40 as the core and PHCPTMA as arms. As mentioned above, by the “bottom-up” method, the drug loading can be readily controlled by adjusting the feed ration of monomer/H40 macroRAFT agent. To complete the exploration of the flexibility of this method, three samples with different HCPT content were prepared with varying the mass feed ratio of HCPTMA/H40 macroRAFT agent (3.5 : 1, 7 : 1, and 14 : 1), and denoted as H40-star-PHCPTMA3.5 (hPC-1), H40-star-PHCPTMA7 (hPC-2), and H40-star-PHCPTMA14 (hPC-3). Figure 3C presents the 1H NMR spectrum of hPC-3, compared with the 1H NMR spectrum of H40 macroRAFT agent in Figure 3B, the new peaks at δ = 7.2-8.0 (d, c, e, f, h), 6.4 (l), 5.3 (i), 4.9 (g), 1.8 (j, b), and 1.4 (a) ppm confirm that the HCPTMA has been well polymerized. In addition, hPC were also characterized by UV-Vis


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(Figure S5), FTIR (Figure S3B), and fluorescence spectroscopy (Figure S6). All these results demonstrate that multi-ployprodrug-arm hyperbranched core hPC have been synthesized successfully. More importantly, as depicted in Table S1, with the increase of the mass feed ratio of HCPTMA/H40 macroRAFT agent, the increasing of the molecular weight of hPC are observed, and the number of HCPT in hPC was tuned from 41 (hPC-1) to 73 (hPC-2), and further to 133 (hPC-3), which is benefit from the flexibility of the “bottom up” approach. It can be predicted that the covalently conjugated nature of HCPT within hPC via an acid-labile ester bond will ensure loading stability, and still with the ability to release HCPT in the acid environment of tumor cells. Finally, multi-ployprodrug-arm hyperbranched core hPC-3 was employed as a hyperbranched macroRAFT agent to mediate the polymerization of zwitterionic monomer MPC to get the hydrophilic shell (PMPC), the obtained multipolyprodrug-arm hyperbranched amphiphiles was denoted as H40-star-(PHCPTMA-bPMPC), hPCM. The multi-ployprodrug-arm hyperbranched core hPC-3 is not soluble in H2O due to its hydrophobicity, while hPCM is readily soluble in H2O. So, the good solubility of hPCM in H2O verifies the core-shell structure with hPC-3 as hydrophobic core and PMPC as hydrophilic shell. The 1H NMR spectrum and the attributions of hPCM in D2O are shown in Figure 3D, which reveal the presence of characteristic signals of PMPC. We did not observe any signals of the hyperbranched polyprodrug core H40-star-PHCPTMA due to its hydrophobic property. In addition, hPCM was also characterized by FTIR (Figure S3B), UVVis (Figure S5), and fluorescence spectroscopy (Figure S6). These results collectively confirm the successful synthesis of the multi-polyprodrug-arm hyperbranched amphiphiles hPCM. It can be imaged that the covalently interconnected core-shell structure will improve the in vivo stability of hPCM, which is beneficial for the extended blood circulation and enhanced accumulation in tumor tissue. Characterization of hPCM Micelles. The size and morphology of the hPCM micelles


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were studies by DLS and TEM. As shown in Figure 4A, the diameter of H40 is determined to be ~ 2.3 nm (PDI = 0.155) in DMSO. By polymerizing prodrug monomer HCPTMA as the arms on the surface of H40, the size of hPC-3 increases to ~ 11.8 nm (PDI = 0.227) in DMSO. After growing hydrophilic PMPC shell on the surface of hydrophobic hPC core, the average size of hPCM further increase to ~ 159 nm (PDI = 0.221) in aqueous solution with a concentration of 1 mg/mL. The stability of hPCM micelles in aqueous media was evaluated by comparing the average size of freshly prepared micelles with that of the micelles stored at room temperature for 50 days. The size of the hPCM micelles (Figure S8) did not change significantly, indicating good stability of hPCM micelles. The surface charge of the hPCM micelles solution was also studied (Figure S9), the result shows that the value of zetapotential is negative (-13.6 mV) in aqueous solution. The TEM image of the hPCM micelles shows a spherical morphology with average diameter of around 75 nm (Figure 4B). This size is slightly smaller than that measured by DLS due to the micelles shrinkage upon dehydration during TEM sample preparation.

Figure 4. (A) DLS curve of H40, hPC-3 (in DMSO), and hPCM (in water). (B) TEM image of hPCM micelles. According to the previous reports, H40 amphiphilic hyperbranched copolymers might exit as unimolecular micelles or multimolecular aggregates above the CAC, depending on the relative block lengths of inner hydrophobic and outer hydrophilic blocks. To investigate the


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aggregation behavior of hPCM in aqueous solution, the critical aggregation concentration (CAC) was monitored by the fluorescence spectroscopy using Nile red as a probe. According to the inflection of the fluorescence spectroscopy (Figure 5A), the CAC value of hPCM is about 46 µg/mL. This result confirms the fact that hPCM unimolecular micelles will aggregate into multimolecular aggregates above CAC in aqueous solution. To further verify the result, hydrodynamic size of hPCM micelles at varying concentrations (range from 1.00.03 mg/mL) were measured by DLS to investigate their aggregation behavior. As shown in Figure 5B, the hydrodynamic size of hPCM in aqueous solution at 1.0 mg/mL is determined to be ~ 159 nm, which is similar to 164 nm of 0.5 mg/mL. More importantly, DLS curves give bimodal distribution with sizes of ~ 152 nm and ~ 30 nm when the concentration of hPCM decreases to 0.25 mg/mL or 0.125 mg/mL. As the concentration of hPCM further decreases to 0.03 mg/mL, the size distribution histogram shows the unimodal distribution with a particle size of ~ 28 nm. According to the previous reports, the size ~ 30 nm is related to the hPCM unimolecular micelles, and ~ 150 nm should be multimolecular aggregates formed from the unimolecular micelles, which is consistent with the CAC result. Based on these results, it is clear that the size of hPCM at high concentration (1.0 mg/mL and 0.5 mg/mL) remains almost constant (~ 150 nm) with relatively stability. With the decrease of the concentration, the multimolecular aggregates will gradually disassembly into unimolecular micelles (~ 28 nm). Although the multimolecular aggregates may dissociate in the blood due to the severe dilution, the size of the resulting stable unimolecular micelles facilitate the accumulation of drug in the tumor sites.


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Figure 5. (A) Critical aggregation concentration (CAC) of hPCM. (B) DLS curve of hPCM aqueous solutions at concentration of 1.0 mg/mL, 0.5 mg/mL, 0.25 mg/mL, and 0.125 mg/mL and 0.03 mg/mL. In Vitro Drug Release. In order to determine the loading content of HCPT, hPCM was stirring in 0.1 N HCl aqueous solutions (0.2 mg/mL) for 72 h to breaking the ester bound completely. Then, the amount of HCPT was determined to be ~ 19.7 wt % by HPLC analysis. To further study the effects of pH values on the HCPT release rates from the hPCM. In vitro drug release studies were carried out under simulated physiological (pH 7.4) and acid acetate buffer (pH 5.4) conditions at 37 °C with shaking. As displayed in Figure 6, the pH value of the medium has a strong effect on the release rate of HCPT from the hPCM, and the release rate of HCPT much higher at a pH of 5.0 than at a pH of 7.4. After 36 h, the cumulative release of HCPT is below 10% in neutral PBS medium (pH = 7.4), which is considerably lower than the release of HCPT (about 48%) in acidic acetate buffer (pH = 5.0). These results identify that the hPCM micelles can remain stable during circulation in the blood stream (pH = 7.4), and can provide a desirable level of drug after the micelles were trapped in endosomes or lysosomes.


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Figure 6. Cumulative release curve of HCPT from hPCM under different pH values (7.4 and 5.0) at 37 °C. Cellular Uptake. For cancer therapy, the uptake of micelles by tumor cells affects the therapeutic efficacy. In order to investigate the cell uptake behavior of the hPCM micelles, fluorescent probe Rhodamin B was conjugated into hPCM to prepare RB-conjugated hPCM. Then, the cell internalization of RB-conjugated hPCM by MCF-7 cells was analyzed by flow cytometry, the results were expressed as the increase of cell fluorescence intensity. As shown in Figure 7A and 7B, the fluorescence signal of Rhodamin B is observed in the cells after 15 min incubation with RB-conjugated hPCM micelles. With the increase of the incubation time (30 min, 1 h, 2 h and 4 h), the relative mean fluorescence intensity of RB-conjugated hPCM micelles pretreated cells obviously increase, which can be attributed to the increased micelles uptake. The flow cytometry analysis result confirms that the hPCM micelles can be effectively internalized by the MCF-7 cells. We then performed CLSM measurement to further investigate the cellular uptake behavior of hPCM micelles. MCF-7 cells were cultured with RB-conjugated hPCM micelles for predetermined time intervals (15 min, 30 min, 1 h, 2 h and 4 h) before observation. After staining the nucleus with Hoechst 33342, the pretreated cells were observed by CLSM. As depicted in Figure 7C, slight fluorescence points are observed in the cytoplasm after short incubation (15 min). With the increase of the incubation


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time (30 min and 1 h), fluorescence signal raise continually in the cytoplasm. When the incubation time was prolonged to 4 h, the red fluorescence of RB-conjugated hPCM micelles can be clearly observed in both cytoplasm and nuclei according to the merged image. These results further demonstrate the effectively internalization of hPCM micelles by the MCF-7 cells.

Figure 7. Cellular uptake behavior of hPCM micelles by MCF-7 cells. (A) Flow cytometry histogram profiles of MCF-7 cells incubated with RB-conjugated hPCM micelles for different time interval (15 min, 30 min, 1 h, 2 h, and 4 h), the untreated cells are used as a control. (B) The relative geometrical mean fluorescence intensities. (C) CLSM images of MCF-7 cells incubated with RB-conjugated hPCM micelles for (a) 15 min, (b) 30 min, (c) 1 h, (d) 2 h, and (e) 4 h. Cell nuclei were stained with Hoechest 33342. In vitro cytotoxicity studies. The in vitro antitumor activity of hPCM micelles at different


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concentrations (0.1-200 µg/mL) was investigated in MCF-7 cells by MTT assay, comparing with free HCPT at 48 h post-treatment. The untreated cells were used as a control. As shown in Figure 8A, the therapeutic efficacy of hPCM micelles is strongly dependent on drug concentration. At low concentrations (< HCPT-equivalent concentration of 50 µg/mL), the cytotoxicity of free HCPT is slightly higher than hPCM micelles. However, with the increase of the HCPT concentration, the hPCM micelles show slightly better anticancer efficiency than free HCPT. This may be partly due to the difficulty of cell uptake of hydrophobic free HCPT by tumor cells, especially at high concentration. Overall, after conjugating to the hPCM, the solubility of HCPT is improved significantly, which facilitates more HCPT to enter the cell and cause fatal damage to tumor cells. In general, most small-molecule anticancer drugs kill tumor cells by activating apoptosis. Here, to investigate whether the death of MCF-7 cells incubating with hPCM micelles is associated

with apoptosis,

the

FITC-Annexin

V/propidium

iodide

(PI)

method was performed. MCF-7 cells were first incubated with hPCM micelles or free HCPT (HCPT-equivalent concentration of 150 µg/mL) for 48 h, and then subjected to FITCAnnexin V/PI staining. The untreated cells were used as a control. The flow cytometry analysis is shown in Figure 8B. After incubation with free HCPT, the ratio of apoptotic cells of the whole MCF-7 is about 35.7%, and the early apoptotic cells take a percentage of 34.54%. After incubation with hPCM micelles, the ratio of apoptotic cells increases to 49.6%, and the majority of them are also the early apoptotic cells. These results clearly show that the hPCM micelles can induce MCF-7 cells apoptosis, and in comparison with free HCPT, the hPCM micelles promote a higher apoptotic rate of MCF-7 cells with the same dose.


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Figure 8. (A) Cell viability of MCF-7 cells against hPCM micelles or free HCPT after culture for 48 h. (B) Flow cytometric analysis for apoptosis of MCF-7 cells treated with hPCM micelles or free HCPT at HCPT-equivalent concentration of 150 µg/mL for 48 h. Lower left, living cells; Lower right, early apoptotic cells; upper right, late apoptotic cells; upper left, necrotic cells. Inserted numbers in the profiles indicate the percentage of the cells present in this area. In vivo pharmacokinetics and biodistribution. It has been proved that nanoparticles with a suitable size (

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