Supramolecular Peptide Therapeutics: Host–Guest Interaction-Assisted Systemic Delivery of Anticancer Peptides

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Dec 2020Supramolecular Peptide Therapeutics: Host–Guest Interaction-Assisted Systemic Delivery of Anticancer Peptides Hua Wang†, Ya-Qiong Yan†, Yu Yi, Zi-Yu Wei, Hao Chen, Jiang-Fei Xu, Hao Wang, Yuliang Zhao and Xi Zhang Hua Wang† Key Laboratory of Organic Optoelectronics & Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084 , Ya-Qiong Yan† CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190. , Yu Yi *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected]. CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190. , Zi-Yu Wei CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190. , Hao Chen Key Laboratory of Organic Optoelectronics & Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084 , Jiang-Fei Xu Key Laboratory of Organic Optoelectronics & Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084 , Hao Wang CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190. , Yuliang Zhao *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected]. CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190. and Xi Zhang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected]. Key Laboratory of Organic Optoelectronics & Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084 https://doi.org/10.31635/ccschem.020.202000283 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail The poor stability of therapeutic peptides in physiological environments hampers their therapeutic efficacy. In this work, a strategy of supramolecular peptide therapeutics (SPT) was proposed for the improvement of the stability and anticancer efficacy of the peptides in vivo. N-Terminal phenylalanine-containing cytotoxic peptides were carried in the cucurbit[7]uril-containing polymer through host–guest interactions between the phenylalanine and cucurbit[7]uril, generating the supramolecular peptide complex with high peptide encapsulation efficiency (> 97%). The formation of the supramolecular peptide complex reserved the biological activities of the peptide, presenting prolonged blood circulation and improved anticancer efficacy. This SPT strategy might provide a cucurbituril- and phenylalanine-functionalized approach for the design and the development of peptide-based pharmaceuticals. Download figure Download PowerPoint Introduction Therapeutic peptides have shown high potentials for treatments of various diseases, including cancers, diabetes, and cardiovascular diseases (CVDs) owing to their advantages of potency, specificity, and low toxicity.1 Although a few peptide-based pharmaceuticals have been marketed, the poor biostability and short plasma half-life are still encountered challenges in the clinical translation of more bioactive peptides into practical use.2,3 To overcome these drawbacks, nanocarrier-based drug delivery systems have been proven to be a promising approach. Nanocarriers such as lipid-based nanoparticles,4 polymeric micelles,5,6 vesicles,7 peptide amphiphiles,8–10 polymer–peptide conjugates,11–14 and inorganic nanoparticles15,16 have been developed for delivery of peptides and proteins, which, however, usually face the problems of low encapsulation efficiency and complicated preparation procedure. Recently, the strategy of supramolecular chemotherapy has attracted increasing interest,17–21 due to the minimization of side effect of the drug oxaliplatin to normal cells, fabricated through the formation of a supramolecular complex with the cucurbit[7]uril (CB[7]) via host–guest interactions. Among macrocycles, the cyclodextrin-based drug delivery systems have been investigated widely22–24 with several formulations such as CALAA-01 and CRXL101 enrolled in clinical trials.25,26 Compared with cyclodextrins, the CB[7] presents a higher maximum binding ability to guest molecules.27 In addition, the CB[7] has a considerable water-solubility, as well as good biocompatibility,28,29 thereby, demonstrating high potentials in pharmaceutical applications. For instance, chemotherapeutic drugs, including cisplatin,30 oxaliplatin,31,32 and others, have been delivered to cancer cells using CB[7]-based molecules. On this basis, supramolecular polymeric chemotherapy enhanced the circulation performance and safety of oxaliplatin via complexation between polyethylene glycol (PEG)-based poly-CB[7] and oxaliplatin.33,34 However, achieving a high therapeutic efficacy with reduced side effects in vivo remains a critical challenge for cucurbituril-based nanocarriers. Besides chemical drugs, recent reports indicated that CB[7] binds with peptides and proteins owing to its high affinity to several aromatic amino acids, especially phenylalanine,35–39 suggesting that the CB[7] might be a promising candidate for peptides- and proteins-based drug delivery. Herein, we report a strategy named the supramolecular peptide therapeutics (SPT) for the enhancement of in vivo efficacious cancer therapy. Our approach was to generate nano-scaled supramolecular peptide complexes from N-terminal phenylalanine (F)-containing cytotoxic peptides and CB[7]-containing copolymers (poly-CB[7]) through host–guest interactions for systemic delivery of anticancer peptides against tumors (Scheme 1). The anticancer peptide was prepared through the standard fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis, in which the natural amino acid F was introduced readily into the N-terminal of the peptide to create a supramolecular peptide complex, expected to possess the following three advantages: (1) an extremely high peptide encapsulation efficiency (> 97%) through a simple mixing procedure in aqueous solution, (2) biological properties of prolonged blood circulation to enhance tumor accumulation of > two-fold with a subsequent improvement of tumor growth inhibiting efficacy, and (3) low toxicities to the hematologic organ systems, including the liver and the kidney. Scheme 1 | The illustration of the formation of supramolecular peptide complex between ploy-CB[7] and F-KLAK. Download figure Download PowerPoint Experimental Method All experimental methods are included in the Supporting Information. Results and Discussion To construct the supramolecular peptide complex, an N-terminal F-containing cytotoxic peptide F-KLAK [sequence: FGGD(KALKALKKALKALK), where the amino acid codes indicated in the bracket represent D-amino acids; Supporting Information Figures S1 and S2], and the poly-CB[7], a copolymer of PEG and CB[7] ( Supporting Information Figure S3), were mixed in equimolar ratio of CB[7] unit/F-KLAK (1∶1) using a vortex mixer stirring with a speed of 2500 rpm for 1 min under ambient conditions (room temperature, 1 atm). The molecular weight (MW), polydispersity index (Đ), and degree of polymerization (DP) for CB[7] of poly-CB[7] were determined to be 70 kDa, 1.60, and 20, respectively, using gel permeation chromatography (GPC). Moreover, a proton nuclear magnetic resonance (1H NMR) spectrometry was performed to investigate the formation of the poly-CB[7]/F-KLAK (PCB/FK) complex with CB[7] unit/F-KLAK = 1∶1 (PCB/FK (1∶1). The peaks of aromatic protons in F-KLAK shifted completely from 7.3–7.4 to 6.4–6.8 ppm after mixing with poly-CB[7], indicating that the peptides were indeed loaded into the CB[7] units in poly-CB[7] (Figure 1a). The benefit of this high encapsulation efficiency was obtained from the strong host–guest interaction between F-KLAK and CB[7], whose binding constant (Ka) was determined to be 2.38 × 106 M−1 using isothermal titration calorimetry (ITC) measurements (Table 1 and Supporting Information Figure S4). Based on the Ka between F-KLAK and CB[7], the encapsulation efficiency of the peptide onto CB[7] moiety was calculated to be 97.2% at 0.5 mM administration concentration of F-KLAK (for more details, refer to "Materials and Methods" section in the Supporting Information), and the corresponding peptide loading content was caculated to be 44.2%. Notably, the binding affinity of the N-terminal F and CB[7] both contributed to the release of high-energy water trapped in the cavity of CB[7] and the ion–dipole interaction between the N-terminal ammonium and carbonyls on the portal of CB[7].38 The enthalpy (ΔH) and entropy (TΔS) changes during the complexation were determined to be −9.24 and −0.55 kcal/mol, respectively, indicating that the formation of the CB[7]/F-KLAK complex was an enthalpy-driven process with less favorable entropy. The binding constant between F-KLAK and poly-CB[7] was determined to be 1.14 × 105 M−1 (Figure 1b and Table 1), which was smaller than that between F-KLAK and CB[7], due to the retardation of the structural remodeling of the nanoparticle. Of note, the PEG did not affect the encapsulation efficiency of F-KLAK, proven by 1H NMR spectra (Figure 1a; Supporting Information Figure S5). The thermodynamic data showed that the binding between poly-CB[7] and F-KLAK resulted in a marked decrease in TΔS, relative to the binding between CB[7] and F-KLAK (−3.16 kcal/mol vs −0.55 kcal/mol; Supporting Information Figures S4 and S6), indicating that the peptides were loaded in poly-CB[7] in a controlled manner. Considering the strong binding affinity of N-terminal F and CB[7], the supramolecular peptide complex exhibited excellent stability in saline without apparent changes in 1H NMR spectra for at least 2 weeks ( Supporting Information Figure S7), compared with those of cyclodextrin-based drug delivery systems, which were usually unstable in physiological saline because of their relatively week binding affinities for guest molecules with a maximum value of 104–105 M−1.40,41 In addition, dynamic light scattering (DLS) measurements were performed to investigate the self-assembly behavior of PCB/FK complexes. We observed remarkably low scattered light intensities of KLAK and F-KLAK in phosphate-buffered saline (PBS; pH = 7.4) at a concentration of 100 μM, indicating that no assemblies occurred ( Supporting Information Figure S8). Meanwhile, poly-CB[7] and PCB/FK complexes presented high scattered light intensities with diameters of approximately 80–130 nm ( Supporting Information Table S1), suggesting the formation of nanosized assembly structures. Notably, the PCB/FK aggregates with diameters ∼ 100 nm were found in Cryo-TEM images ( Supporting Information Figure S9). Compared with the poly-CB[7], increased in size and zeta-potential for PCB/FK complexes occurred possibly because of the conjugation of positively charged peptides onto the surface of the nanoparticles consisting of poly-CB[7]. Moreover, as the KLAK peptide was able to form alpha-helical structure upon interaction with biological membranes,42 the circular dichroism (CD) spectrometry was carried out to study the changes in secondary structure of the supramolecular peptide complex. Our results showed that the complexation with poly-CB[7] had limited effects to the secondary structure of the F-KLAK against liposomes as a model of membrane ( Supporting Information Figure S10), suggesting that the poly-CB[7] retained the bioactivity of the loaded peptides. Figure 1 | The host–guest interaction between poly-CB[7] and F-KLAK characterized by (a) 1H NMR (400 MHz, D2O, 25 °C) with a peptide concentration of 0.5 mM and (b) ITC measurement (20 mM phosphate buffer, pH = 7.4, 37 °C). The PCB/FK (1∶1) was prepared with a molar ratio of CB[7] unit/F-KLKA as 1∶1. Download figure Download PowerPoint Table 1 | Thermodynamic Parameters of the Binding of CB[7] and Poly-CB[7] with F-KLAK at 20 mM Phosphate Buffer (pH = 7.4, 25 °C). Sample Ka (105 M−1) ΔH (kcal mol−1) TΔS (kcal mol−1) ΔG (kcal mol−1) CB[7] and F-KLAK 23.80 ± 0.97 −9.24 −0.55 −8.69 Poly-CB[7] and F-KLAK 1.14 ± 0.05 −10.60 −3.16 −7.44 Prior to the investigations of the in vivo anticancer efficacy of the PCB/FK supramolecular peptide complexes, the influence of the KLAK peptide on the cytotoxicity of in vitro cancer cells was studied by viability cell counting Kit-8 (CCK-8) assay, after the introduction of the amino acid F to the N-terminal. Surprisingly, the incorporation of F intensively increased the cytotoxicity of KLAK peptide for colorectal cancer (HCT116) cells, leading to a decrease in the 50% inhibitory concentration (IC50) from 162.4 μM for KLAK to 15.5 μM for F-KLAK (Figure 2). The reason for this striking reduction in the IC50 might be due to the improvement of the membrane-rupturing capability of the F-KLAK peptide as a result of the introduction of the hydrophobic F. To demonstrate this hypothesis, we performed ITC measurements on a liposome membrane model, prepared from l-alpha-phosphatidyl-l-serine and 1,2-distearoyl-sn-glycero-3-phosphocholine mixture. When KLAK peptides were titrated into the negatively charged liposomes, an exothermic curve was observed over the whole time course of the isothermal titration, reflecting the electrostatic interaction (Figure 3a and Table 2), compared with F-KLAK, which upon the initial addition into the liposomes, exhibited a smaller exothermic enthalpy (–18.2 kcal/mol vs −31.8 kcal/mol, respectively; Figure 3b). This F-KLAK observation might also be attributable to the insertion of the residual F into the membrane, driven by an endothermic hydrophobic interaction. Interestingly, a small endothermic process also occurred before the end point of titration, implying the solubilization or rupture of the liposomes by F-KLAK.43,44 This explanation was confirmed by DLS measurements ( Supporting Information Table S2). An addition of F-KLAK to the liposomes increased their size significantly from 200 to 3000 nm, while FGG and KLAK had limited influence on the size of the liposomes. Besides, a membrane fluorescent dye, Nile red, was also encapsulated in the liposomes to monitor the membrane rupture. The principle was that the leakage of Nile red from the liposomes, arising from the membrane rupture, would lead to an ultimate decrease in the total fluorescence intensity. As shown in Figure 3c, F-KLAK induced the more intensive leakage of Nile red from the liposomal nanoparticle than KLAK, revealed by differential decrease in fluorescence intensity (35% vs 24%), which indicated a better membrane-rupturing ability for F-KLAK. Meanwhile, the FGG presented a negligible effect on the liposomal membrane damage, suggesting the importance of the synergetic effect between F and KLAK peptide. Figure 2 | Comparison studies of anticancer activities for F-KLAK and KLAK upon cultured (a) HCT116 cells and (b) colorectal normal (NCM460) cells. The cells were incubated with KLAK and F-KLAK for 48 h, respectively, followed by determination of the viability using CCK-8 assay. Results are expressed as mean and standard deviation (n = 6). Download figure Download PowerPoint Table 2 | Thermodynamic Parameters of the Binding of Peptides with Liposomes Derived from the ITC Curves in Figure 3a and 3b. Sample ΔH (kcal mol−1) TΔS (kcal mol−1) ΔG (kcal mol−1) KLAK −31.8 −21.9 −9.28 F-KLAK ΔH1 −18.2 TΔS1 −6.49 ΔG1 −11.7 ΔH2 2.35 TΔS2 12.6 ΔG2 −10.3 Afterward, the influence of F-KLAK on the anticancer activity after complexation with poly-CB[7] was examined. The CB[7] complexation reduced the cytotoxicity of F-KLAK for HCT116 cells because of the shielding of the F moiety (Figure 4a and Supporting Information Figure S11). Fortunately, the multivalent effect induced by the poly-CB[7] improved the cytotoxicity of PCB/FK complexes substantially, which was close to that of the naked F-KLAK, especially, when an equimolar ratio of PCB/FK (1∶1) was used (Figure 4b and Supporting Information Figure S12). Noticeably, the anticancer activity of the PCB/FK complex was ascribed to the peptide instead of poly-CB[7] due to the negligible cytotoxicity of poly-CB[7] ( Supporting Information Figure S13). In addition, the anticancer efficacy of the PCB/FK complex decreased with an increase in the feeding ratio of CB[7] unit/F-KLAK (Figure 4b), because of the shielding effect of the excess poly-CB[7] to the peptide. This assumption was supported by the fact that the cell internalization process decreased with an increase in the CB[7] unit/F-KLAK ratio in PCB/FK complexes ( Supporting Information Figure S14). As the anticancer activity of KLAK peptides was derived from the disruption of the mitochondria in tumor cells, the ability of PCB/FK complexes to deliver F-KLAK peptides to mitochondria was studied further by observing colocalization of the red Cy5-labeled F-KLAK peptides fluorescence with the mitochondrial-targeted green MitoTracker (MTG) fluorescence using confocal microscopy. As shown in Figure 5, after incubations with the Cy5-labled peptides and PCB/FK complexes for 24 h, the HCT116 cells treated with F-KLAK presented better overlap between mitochondrial MTG-labeled (green) and Cy5-labled F-KLAK peptides (red). Also, a higher Pearson's correlation coefficients quantified from colocalization analyses ( Supporting Information Figure S15) was observed with F-KLAK than the KLAK treated, indicating better mitochondrion-targeting abilities of F-KLAK. Simultaneously, PCB/FK (1∶1) presented similar overlaps of two the colors in confocal microscopic images and Pearson's correlation coefficients (Figure 5 and Supporting Information Figure S15), suggesting that PCB/FK (1∶1) could deliver F-KLAK peptides efficiently to mitochondria in cancer cells. Notably, we noticed lower Pearson's correlation coefficients for PCB/FK (2∶1), and much lower for PCB/FK (3∶1). Hence, the excessive shielding by poly-CB[7] might have contributed to the reduced cytotoxicity, compared with PCB/FK(1∶1). Collectively, the anticancer activity of PCB/FK complex decreased with increased PCB ratio. Therefore, the results of the in vitro anticancer efficacy and mitochondrion colocalization fluorescence studies suggested that PCB/FK (1∶1) and PCB/FK (2∶1) were potential candidates for efficient cancer therapy, and thus, were applied in further evaluations in vivo. Figure 4 | Comparison studies of anticancer activities for (a) F-KLAK and CB[7]/F-KLAK complex with the molar ratio of 1∶1 {CB[7]/FK (1∶1)}, as well as (b) F-KLAK and PCB/FK complexes with different CB[7] unit/F-KLAK molar ratios (1∶1, 2∶1, and 3∶1) upon cultured HCT116 cells. The cells were incubated with each drug sample for 48 h, followed by determination of cell viability using CCK-8 assay. Results are expressed as mean and standard deviation (n = 6). Download figure Download PowerPoint The plasma clearance rate of the PCB/FK complex was studied to evaluate the stability of the peptide in blood circulation. Unlike the naked peptide with a fast clearance rate (18% of injected dose per mL plasma remained after 30 min), PCB/FK (1∶1) and PCB/FK (2∶1) presented significant slower clearance rates (29% and 32% of injected dose per mL plasma remained after 30 min, respectively; Figure 6a), indicating that the formation of supramolecular peptide complex improved the stability of the carried peptide. Furthermore, the two PCB/FK complexes showed significantly enhanced tumor accumulations, compared with the naked F-KLAK [PCB/FK (1∶1) = 2.4-fold and PCB/FK (2∶1) = 2.8-fold enhancement; Figure 6b], attributable to their prolonged blood circulation properties. Additionally, compared with the naked F-KLAK, the PCB/FK complexes presented similar distributions in the kidney, but slightly increased accumulations in the other main organs, especially, with PCB/FK (2∶1) (Figure 6b). In other words, PCB/FK significantly improved the stability of the peptide in blood circulation and enhanced the tumor accumulations. Considering the results of the encapsulation efficiency, anticancer activity, plasma clearance rate, and tumor accumulation, PCB/FK (1∶1) was the best candidate to be employed in the evaluation of in vivo toxicology and anticancer efficacy studies. Figure 6 | In vivo performances of PCB/FK complexes. (a) Blood clearance profiles of the FITC-labeled F-KLAK and its supramolecular complexes (PCB/FK (1∶1) and PCB/FK (2∶1)) (n = 3, the *p < 0.05 was considered as statistically significance between the naked F-KLAK with both the PCB/FK (1∶1) and PCB/FK (2∶1)). (b) Biodistribution of the Cy5-labeled F-KLAK and its supramolecular complexes [PCB/FK (1∶1) and PCB/FK (2∶1)] at 24 h post injection (n = 3, *p < 0.05). (c) Tumor growth profiles and (d) changes in body weight of the treated mice during anticancer therapy. The saline, KLAK, F-KLAK, and PCB/FK (1∶1) were intravenously administrated into the tail vein of mice (10 mg/kg peptide/injection, one injection for every 2 days, with a totally of 10 injections). Results are expressed as mean and standard deviation (n = 5, *p < 0.05). Download figure Download PowerPoint With the identification of the optimum conditions for the supramolecular peptide complex formation and high encapsulation efficiency to fabricate PCB/FK (1∶1), we verified the biological safety of our SPT strategy in healthy mice, divided into 2 × 4 groups (n = 3). We first performed systemic administrations of the following drugs in the four mice groups: Group 1 = saline control, Group 2 = KLAK, Group 3 = F-KLAK, and Group 4 = PCB/FK (1∶1), with 10 mg/kg peptide/injection at 24 h intervals for 2 injections. Next, in vivo toxicology studies were carried out at two time points, 24 h and 7 days after the final injection. Our results showed no apparent changes in the plasma levels of the hematologic indexes (red blood cell count, white blood cell count, platelet count, and hemoglobin); liver functions (alanine aminotransferase, aspartate aminotransferase, total protein, and total bilirubin); and kidney functions (urea, creatinine, and uric acid) for all tested samples at the two time points after the injections (Figure 7 and Supporting Information Table S3), indicating the biological safeness of F-KLAK and PCB/FK (1∶1) under the tested experimental conditions. Figure 7 | The plasma levels determination of (a) red blood cell (RBC), (b) white blood cell count (WBC), (c) alanine aminotransferase (ALT), and (d) creatinine (CREA) of treated mice after administrations of saline, KLAK, F-KLAK, and PCB/FK (1∶1). Results are expressed as mean and standard deviation (n = 3). Download figure Download PowerPoint Moreover, the anticancer efficacy of the supramolecular peptide complex was investigated on the subcutaneous HCT116 tumor-bearing mouse model. Through intravenous administrations of the saline, KLAK, F-KLAK, and PCB/FK (1∶1) with a dosage of 10 mg/kg peptide/injection for a total of 10 injections (one injection for every 2 days), the PCB/FK (1∶1), significant suppression of the tumor growth in the drug-treated mice was apparent, demonstrating ∼ tumor growth increase in the tumor size on after the first compared with the tumor growth in the saline, KLAK, and F-KLAK, which showed as high as and (Figure indicating an improved tumor growth of the PCB/FK (1∶1) supramolecular peptide complex. Meanwhile, during the no body weight was observed in the treated mice (Figure suggesting negligible systemic for the peptides and PCB/FK (1∶1) under the experimental conditions. In addition, no was found in the main of the treated mice in ( Supporting Information Figure after followed by and and light which confirmed further the safeness of PCB/FK (1∶1) during the we that this SPT strategy tumor growth while normal physiological activities in the mice model. Figure 5 | of Cy5-labled KLAK, F-KLAK, and PCB/FK (1∶1) after 24 h against cultured HCT116 cells 10 The images showed Cy5-labled peptides mitochondria by MitoTracker and their 20 Download figure Download PowerPoint We have presented a strategy of supramolecular peptide therapeutics to cancer therapy. This enhanced for therapeutic peptides in physiological environments by the formation of supramolecular peptide complex via host–guest interactions of the N-terminal phenylalanine and The supramolecular peptide complex with a high peptide encapsulation efficiency several including slower blood enhanced tumor accumulation, and for tumor growth a promising approach for peptide-based cancer therapeutics with reduced side this strategy could be applied to and deliver therapeutic peptides the the Considering the for peptide-based pharmaceuticals for clinical SPT might provide a and potential for clinical of bioactive peptides and Figure 3 | of membrane rupture for KLAK and F-KLAK characterized by using ITC measurements KLAK and F-KLAK, pH = 7.4, 37 °C) and (c) fluorescence of Nile red encapsulated liposomes nm, nm, 25 °C). Download figure Download PowerPoint Supporting Information Supporting Information of no of to Information This was supported by the of and Technology of and and the National of and The to Wang (NCNST), (NCNST), Zhang of Chemistry, of and Wang of Chemistry, of for their the experimental Zhao Nanocarriers for in Therapeutics: and 20, Chen Delivery of In and In Zhao Xu Zhang of as Zhao Chen for at

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