Mussel-Derived, Cancer-Targeting Peptide as pH-Sensitive Prodrug

Jun 13, 2019 - In this work, we prepared a novel cancer chemotherapeutic nanocarrier through the self-assembly of a mussel-derived, cancer-targeting ...
0 downloads 0 Views 8MB Size
Download PDF
Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 23948−23956

www.acsami.org

Mussel-Derived, Cancer-Targeting Peptide as pH-Sensitive Prodrug Nanocarrier Yue Ma,†,‡ Peiyan He,‡ Xiaohua Tian,† Guanglei Liu,† Xiaowei Zeng,§ and Guoqing Pan*,† Institute for Advanced Materials, School of Materials Science and Engineering and ‡School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, China § School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Guangzhou 510275, China

Downloaded via NOTTINGHAM TRENT UNIV on July 19, 2019 at 03:12:08 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

ABSTRACT: In this work, we prepared a novel cancer chemotherapeutic nanocarrier through the self-assembly of a musselderived, cancer-targeting peptide with a pH-sensitive conjugation of antitumor drugs. The biomimetic peptide was designed with a fluorescent molecule fluorescein isothiocyanate for imaging, a RGD sequence for cancer-targeting and tetravalent catechol groups for dynamic conjugation of the antitumor drug bortezomib via pH-cleavable boronic acid−catechol esters. Our study demonstrated that the peptide-based prodrug nanocarrier dramatically the enhanced specific cellular uptake and cytotoxicity toward human breast cancer cells in vitro in comparison with free drug and nontargeting control nanoparticles. Likewise, the prodrug nanocarrier showed improved therapeutic efficacy and low systematic toxicity in vivo. Considering highly biomimetic nature of the peptide-based nanocarriers, rapid drug release from the dynamically conjugated prodrugs, and convenience of introducing cancer-targeting activity onto this nanosystem, we believe our work would provide new ideas for the development of intelligent and biocompatible drug delivery systems to improve the chemotherapy efficacy in clinic. Furthermore, the pH-sensitive drug conjugation mechanism on peptide-based nanocarriers would provide a hint for the exploitation of dynamic prodrug strategies and the development of highly biocompatible nanocarriers using biogenic materials, e.g., the proteinogenic nanomaterials decorated with drugs through dynamic covalent chemistry. KEYWORDS: cancer therapy, dynamic covalent bond, bortezomib, mussel-derived peptide, prodrug

■

their composites.5,8−12 Although extensive efforts have been made to design novel nanocarriers and integrate multiple functions to achieve targeted cancer cell recognition and controlled drug release, the potential risks of these nanomaterials and their degradation products is still a fundamental problem hard to be well defined. First, these synthetic polymers or inorganic nanoparticles are xenobiotic compounds (or nonbiological foreign chemicals). In addition to the biotoxicity that could be determined in short-term clinical trials,13 their potential harm to human body from a long-term perspective (e.g., genes and heredity) should not be overlooked. Second, most of the degradation products would cause the disturbances of the acid−base balance or excessive

INTRODUCTION Chemotherapy is one of the mainstream systemic treatments for cancers.1 However, it suffers from serious toxic side effects on normal cells and tissues.2 For the past decades, nanomaterials have emerged as very promising drug carriers to improve the accuracy and efficacy of chemotherapy during cancer treatments.3 To achieve an accurate and efficient attack on cancers, nanomaterial-based drug carriers could be endowed with desirable properties like the ability to target cancer cells, enhanced permeability and retention effect, and controlled drug release, etc.4−6 Obviously, the prosperity of nanomaterialbased cancer therapy profits from the rapid development in nanoscience and material engineering, which created more opportunities to design multifunctional drug delivery systems.7 Currently, the most widely studied nanomaterials for antitumor drug delivery mainly focus on the use of biodegradable synthetic polymers, inorganic nanoparticles, or © 2019 American Chemical Society

Received: May 23, 2019 Accepted: June 13, 2019 Published: June 13, 2019 23948

DOI: 10.1021/acsami.9b09031 ACS Appl. Mater. Interfaces 2019, 11, 23948−23956

Research Article

ACS Applied Materials & Interfaces

Figure 1. (a) Structural formula of the mussel-derived cancer-cell-targeting peptide and the dynamic conjugation of antitumor drug BTZ with pHresponsiveness. (b, c) Schematic illustration of the peptide-prodrug-based nanoparticle with cancer cell targeting and pH-sensitive drug release property.

with mannitol, which could suppress nonspecific BTZ binding to proteins, reduce hepatic clearance from blood, and exhibit acidic-enhanced pharmacological activity. However, it is worth mentioning that Velcade lacks targeting mechanism to be transported to tumor tissues effectively, thus exhibiting less effectiveness against solid tumors in vivo.27,28 Therefore, rational combination of prodrug approaches with nanomaterial-based drug delivery systems is a promising method that could synergistically address the serious side effects of antitumor drugs and improve the efficacy of cancer chemotherapy. According to the above considerations, we propose to prepare a novel cancer chemotherapeutic nanocarrier based on the self-assembly of biomimetic peptides with dynamically conjugated antitumor prodrugs. As is known, peptide-based nanocarriers are highly biocompatible as compared to synthetic polymers or inorganic nanoparticles. Apart from the biogenic nature of peptides, their degradation products by plasma proteases are merely amino acids (AAs), which are the essential nutrients for human body.29−31 In addition, peptides have conveniences for the conjugation of antitumor drugs and cancer-targeting motifs. First, the side chains of AAs (e.g., carboxyl or amino groups) enable direct chemical conjugation to obtain peptide-based prodrugs.32,33 Second, the cancertargeting ability of peptide-based nanoparticles could be easily achieved by introducing specific sequences with tumor-

accumulation of trace elements in localized tissues, which could also induce chronic inflammatory response in vivo.14−16 In this context, the exploitation of new nanocarriers with lower potential biological risks and improved efficacy of chemotherapy remains an enormous challenge in medical oncology, and this is also of great significance in biomaterial science. The intrinsic reason of serious side effects in chemotherapy is the high systemic toxicity of antitumor drugs but lack of tumor selectivity. The active sites of drugs could interact with cancer cells as well as the normal cells, thus deteriorating the chemotherapeutic efficacy and leading to side effects. Besides nanomaterial-based carriers for targeting drug delivery, the prodrug approach has also been followed in the past decades for the purpose of improving chemotherapy.6,17,18 Prodrugs are drug-derived chemicals with low or no pharmacological activity. They could undergo biotransformation to a therapeutically active metabolite in vivo.19−21 Reversible blocking of the active sites of drugs by means of cancerspecific characteristics (e.g., weakly acidic pH or the overexpression of tumor-associated biomolecules) could obtain prodrugs with tumor-specific pharmacological activity, thus showing promise for improving chemotherapy efficiency and reducing side effects.22−26 For example, the commercialized anticancer agent Velcade is a prodrug complex of bortezomib (BTZ, antitumor drug) and mannitol. The active boric acid group of BTZ can form a pH-sensitive dynamic covalent bond 23949

DOI: 10.1021/acsami.9b09031 ACS Appl. Mater. Interfaces 2019, 11, 23948−23956

Research Article

ACS Applied Materials & Interfaces

Figure 2. (a, b) ESI-MS spectrum of the mussel-derived peptide PEP-RGD and PEP-RGE. (c, d) HPLC profiles of PEP-RGD and PEP-RGE with high purity (>95%).

■

targeting activity.34−38 In this work, we synthesized a fluorescently labeled biomimetic peptide PEP-RGD (FITC(DOPA)4-G5-RGDS) composed of a cancer-targeting sequence RGDS at the C-terminus, a non-bioactive quintuple glycine G5 spacer, a tetrapeptide (DOPA)4 with catechol groups, and a fluorescent molecule fluorescein isothiocyanate (FITC) for cell imaging at the N-terminal end (Figure 1a). The RGD peptide was used as the model ligand for tumor targeting due to the overexpressed αvβ3 integrin on tumor cell membranes.39 DOPA is a catecholic amino acid 3,4-dihydroxyL-phenylalanine that is abundant in mussel secreted proteins.40−42 The catechol groups in DOPA can bind with boronic acid (BA) through the formation of pH-sensitive catechol/BA dynamic covalent ester bonds.43−45 In this case, the mussel-derived peptide PEP-RGD could react with BAcontaining antitumor drug BTZ to obtain a pH-cleavable peptide-BTZ prodrug BTZ-PEP-RGD. The amphiphilic BTZPEP-RGD could be readily assembled to nanosized particles with RGD motifs on the outer layer (Figure 1b). Besides the cancer-targeting ability of the BTZ-PEP-RGD nanoparticles, here the dynamically conjugated prodrug approach with pHresponsiveness is expected to show faster drug release properties in cells as compared to traditional covalent conjugation. Under neutral biological condition, stable catechol/BA esters in the BTZ-PEP-RGD nanoparticles could reduce premature drug leakage. Upon uptake through endocytosis, the weakly acidic environment of endo/lysosome (pH 5.0−6.0)46 will trigger a rapid dissociation of catechol/BA ester to release free BTZ (Figure 1c), thus enhancing the pharmacological activity and chemotherapeutic efficacy. We anticipate that the highly biomimetic nature of this peptidebased nanocarriers, the rapid responsiveness of dynamically conjugated prodrug, and the simplicity for introducing cancertargeting activity in this work would provide a hint for the development of new drug delivery systems with improved chemotherapy efficacy both in vitro and in vivo.

RESULTS AND DISCUSSION Synthesis of Mussel-Derived Peptides. The musselderived, cancer-targeting peptide PEP-RGD (FITC-(DOPA)4G5-RGDS) and the nontargeting control peptide PEP-RGE (FITC-(DOPA)4-G5-RGES) were synthesized by a standard Fmoc-based solid-phase peptide synthesis strategy.47,48 To reduce the possibility of catechol oxidation during synthesis, acetonide-protected Fmoc-DOPA(acetone)-OH was used to introduce the DOPA amino acids into the peptide sequences. To facilitate fluorescence labeling, the peptides were also capped with 6-aminocaproic acid at the N-terminal for FITC conjugation. The biomimetic peptides were then characterized with electrospray ionization mass spectrometry (ESI-MS). As shown in Figure 2a, the monoisotopic ion peak [M + 2H]2+ of PEP-RGD was detected at m/z 969.8 (monoisotopic mass of 1937.9 Da), which is in good agreement with the calculated mass of FITC-(DOPA)4-G5-RGDS. Meanwhile, the monoisotopic ion peaks [M + 2H]2+ and [M + H]+ of PEP-RGE were found at 977.0 and 1952.6, which matched perfectly the theoretical molecular weight of FITC-(DOPA)4-G5-RGES (1951.93 Da). These results, together with the high peptide purity (>95%) in high-performance liquid chromatography (HPLC) profiles (Figure 2b), confirmed the successful synthesis of the predesigned mussel-derived peptides. Prodrug Nanoparticle Preparation and Drug Release. The two biomimetic peptides PEP-RGD and PEP-RGE were then used to conjugate with BTZ to prepare the peptide-BTZ prodrugs BTZ-PEP-RGD and BTZ-PEP-RGE, respectively. The peptide-BTZ prodrugs could from nanoparticles with small size (173 ± 34 nm) in phosphate buffer saline (PBS) due to their amphiphilic properties (Figure 3a,b). HPLC analysis revealed that the final BTZ loading capacity (wt %) of the BTZ-PEP-RGD and BTZ-PEP-RGE nanoparticles were 37.4 and 36.7%, respectively. This result indicated a high drug loading efficiency, such that the DOPA-containing peptides could conjugate approximately three BTZ molecules per peptide molecule. To show the pH-sensibility of peptide23950

DOI: 10.1021/acsami.9b09031 ACS Appl. Mater. Interfaces 2019, 11, 23948−23956

Research Article

ACS Applied Materials & Interfaces

BTZ prodrug, the BTZ release performances at different pH values from the groups of BTZ-PEP-RGD and BTZ-PEP-RGE nanoparticles were carried out (Figure 3c). To mimic the normal physiological condition, the tumor microenvironment, and subcellular endosome, buffers with pH values at 7.4, 6.5, and 5.0 were used for drug release experiments, respectively. As expected, the two peptide-BTZ prodrug nanoparticles showed a relatively slow release with less than 20% BTZ release at pH 7.4, while approximately 80% of the BTZ could be rapidly released at pH 5.0. This dramatically increased BTZ release in acidic condition confirms the advantages of pHcleavable catechol−boronic acid esters for prodrug design, which could inactivate BTZ and suppress drug permeability in normal tissues to reduce side effects. Moreover, upon uptake by tumor cells, the prodrug nanoparticles would allow endosome-triggered rapid drug release and subsequently accelerate the BTZ-mediated apoptosis of tumor cells. In Vitro Cellular Uptake. RGD is a cell-adhesive peptide sequence with high specificity toward integrin αvβ3 for mediating interactions between cells and components of the extracellular matrix.49−51 The αvβ3 integrin is expressed at relatively lower levels in normal cells, but it is always highly expressed in many types of cancer cells.39 Thus, the RGD peptide has been well-documented as a molecular marker for

Figure 3. (a, b) Transmission electron microscopy (TEM) images of the BTZ-PEP-RGD and BTZ-PEP-RGE prodrug nanoparticles. Scale bar is 500 nm. (c) In vitro BTZ release profiles from the BTZ-PEPRGD and BTZ-PEP-RGE prodrug nanoparticles (0.5 mg/mL) at different pH values (pH 5.0, 6.5, and 7.4).

Figure 4. (a, b) Confocal laser scanning microscopic (CLSM) images of the HEK 293 and MDA-MB-231 cells incubated with the BTZ-PEP-RGD nanoparticles, BTZ-PEP-RGE nanoparticles, and BTZ-PEP-RGD nanoparticles in the presence of RGDS peptide as a competitor. Scale bar is 50 μm. (c, d) Flow cytometric analysis and quantitative results of the cells treated with BTZ, peptide, and the nanoparticles. 23951

DOI: 10.1021/acsami.9b09031 ACS Appl. Mater. Interfaces 2019, 11, 23948−23956

Research Article

ACS Applied Materials & Interfaces

Figure 5. (a) In vitro viability of the HEK 293 and MDA-MB-231 cells after 24 h incubation with the mussel-derived peptides, the peptide-BTZ prodrug nanoparticles, and free BTZ. The untreated cells with 100% viability were used as control. Statistically significant differences are indicated by *p < 0.01 as compared with all others. (b) The CLSM live/dead cell images of the MDA-MB-231 cells in the presence of peptides, free BTZ, and peptide-BTZ prodrug nanoparticles with equivalent BTZ concentration of 100 nM. Scale bar is 200 μm. (c) Flow cytometric results after incubating MDA-MB-231 cells with peptide PEP-RGD, free BTZ, BTZ-PEP-RGE nanoparticles, and BTZ-PEP-RGD nanoparticles (equivalent BTZ concentration of 100 nM) for 24 h.

between the HEK 293 and MDA-MB-231 cells. Moreover, the two cell lines treated with the nontargeting BTZ-PEP-RGE nanoparticles showed a significantly weaker fluorescence, which was only quarter that in the MDA-MB-231 cells treated with the BTZ-PEP-RGD nanoparticles. These results demonstrated the advantage of the BTZ-PEP-RGD nanoparticles with enhanced uptake by the αvβ3-overexpressed tumor cells. To further confirm the tumor-targeting ability of the BTZPEP-RGD nanoparticles, the cells preincubated with free RGDS peptides were used for cellular uptake analysis. In this case, the αvβ3 integrin on cell membrane will be blocked by the binding of free RGDS peptides, resulting in the attenuated internalization of nanoparticles. As expected, the green fluorescence in MDA-MB-231 cells exhibited a significant decrease upon preincubation with RGDS peptide. Conversely, the fluorescence in the HEK 293 cells showed negligible change regardless of the media with or without the free RGDS peptide. These results clearly suggested that the RGD motifs

targeted cancer imaging and therapy. In our design, the combination of FITC and RGD motifs in the BTZ-PEP-RGD prodrug would endow the nanosystem with specific tumor cell uptake and self-imaging properties. Cellular uptake behavior of the nanoparticles was examined on two different cell lines (Figure 4), i.e., the αvβ3-overexpressed MDA-MB-231 cells (human breast cancer cells) and the αvβ3 negative HEK 293 cells (human embryonic kidney cells). Nontargeting BTZPEP-RGE nanoparticles were used as a control. After 4 h of incubation with the BTZ-PEP-RGD nanoparticles, the αvβ3overexpressed MDA-MB-231 cells showed a significantly stronger green fluorescence than the HEK 293 cells did. Flow cytometry analysis was then used for the quantitative characterization of the fluorescent intensity in the cells. The results indicated that the intracellular fluorescence in the MDA-MB-231 cells was 4-fold higher than that in the HEK 293 cells. In contrast, fluorescence intensity in the group of BTZ-PEP-RGE nanoparticles had no significant difference 23952

DOI: 10.1021/acsami.9b09031 ACS Appl. Mater. Interfaces 2019, 11, 23948−23956

Research Article

ACS Applied Materials & Interfaces

Figure 6. (a) Pharmacokinetics of the BTZ drug in blood system after free BTZ, BTZ-PEP-RGE nanoparticles, and BTZ-PEP-RGD nanoparticles were injected into the tumor-bearing mice. The time-dependent changes of (b) tumor tissue sizes and (c) body weights of the mice after treatment (*p < 0.01 and **p < 0.001). (d) Tumor tissue sections stained with hematoxylin and eosin (H&E) after 20 days of chemotherapy. The tumor tissue in group of BTZ-PEP-RGD showed significant tumor necrosis as compared to others.

free BTZ and the two nanoparticles was analyzed by flow cytometry. The vital, early apoptotic, late apoptotic, and dead cells in different groups were differentiated and are shown in Figure 5c. Likewise, the BTZ-PEP-RGD nanoparticles caused the most significant apoptotic characteristics as compared to other groups, and almost all of the MDA-MB-231 cells showed early or late apoptotic characteristics. Undoubtedly, the significantly enhanced cancer cell inhibition of the BTZPEP-RGD nanoparticles mainly benefited from the tumortargeted cell uptake and subsequently endo/lysosome-triggered BTZ release. The above results also indicated the potential of our nanosystem for selective and efficient killing of the αvβ3overexpressed tumor cells than normal cells in vivo. In a word, our mussel-inspired peptide-BTZ prodrug nanoparticles enable tumor-targeting cellular uptake and rapid BTZ release at endo/ lysosome with a weak acidic environment, which would facilitate the rapid and specific inhibition of tumor cells as well as the reduction of drug side effects in clinical chemotherapy. In Vivo Antitumor Efficacy. With the above positive results in vitro, we then carried out animal experiments to examine the in vivo BTZ delivery and antitumor activity of the nanosystem in nude mice model with the MDA-MB-231 cancer xenograft.53 The in vivo pharmacokinetic profiles were first evaluated. As shown in Figure 6a, the BTZ levels in blood were eliminated readily after the administration of free BTZ, exhibiting a very short circulation time. In contrast, the two peptide-based prodrug nanoparticle groups showed significantly longer blood circulation times lasting for 24 h. These results implied that the prodrug nanoparticles with improved circulation in the bloodstream would facilitate BTZ accumulation at the tumor sites. The in vivo antitumor efficacy of our nanocarriers was then investigated by monitoring the changes of tumor sizes during chemotherapy. After 20 days of treatment, the groups of BTZ-PEP-RGD and BTZ-PEP-RGE nanoparticles showed a higher antitumor efficiency than the free BTZ, PEP-RGD peptide, and the saline-treated groups (Figure 6b). This result was in perfect agreement with the

on the BTZ-PEP-RGD nanoparticles are the key factors of tumor cell recognition, further confirming the targeting ability of the BTZ-PEP-RGD nanoparticles toward the αvβ3-integrinoverexpressed tumor cells. In Vitro Antitumor Activity. To show the superiority of acid-sensitive BTZ release and tumor-targeting cellular uptake in our system, in vitro cytotoxicity of the mussel-derived peptides and nanoparticles was then quantitatively examined by thiazolyl blue tetrazolium bromide (MTT) assay (Figure 5a).52 Before conjugation with BTZ drug, the two musselderived peptides exhibited negligible cytotoxicity against the HEK 293 and MDA-MB-231 cells in a wide range of concentrations, indicating the highly biomimetic nature of our mussel-derived peptides. As expected, the cell inhibitions in the BTZ-PEP-RGE and BTZ-PEP-RGD nanoparticle groups showed BTZ-concentration-dependent properties. Nevertheless, no significant difference of the HEK 293 cell viability between the groups of BTZ-PEP-RGE and BTZ-PEPRGD nanoparticles could be observed. On the contrary, the viability of the MDA-MB-231 cells showed completely different results after treatment with BTZ-PEP-RGE and with BTZ-PEP-RGD nanoparticles. For example, at the equivalent BTZ concentration of 100 nM, the MDA-MB-231 cell viability decreased dramatically to only 12% in the BTZPEP-RGD group, while it remained at around 42% in the BTZ-PEP-RGE group. Note that under the same condition, the HEK 293 cell viability was around 55−58% for the groups of free BTZ, BTZ-PEP-RGD, and BTZ-PEP-RGE nanoparticles, suggesting the tumor-preferred cell inhibition of the BTZ-PEP-RGD nanoparticles. To obtain more a intuitive understanding of the cancer cell inhibition efficacy, the MDA-MB-231 cells were incubated with peptides, free BTZ, and nanoparticles for 24 h and then analyzed with a Dead/Live Cell Staining Kit. In line with the results obtained by MTT assay, the BTZ-PEP-RGD nanoparticles showed the highest cancer cell killing ability (Figure 5b). Furthermore, the MDA-MB-231 cell apoptosis induced by 23953

DOI: 10.1021/acsami.9b09031 ACS Appl. Mater. Interfaces 2019, 11, 23948−23956

Research Article

ACS Applied Materials & Interfaces

chloride resin (2.0 mmol), Fmoc-6-aminocaproic acid (AcA), and DIPEA (5 mmol) were dissolved in 20 mL of DCM and then stirred for 4 h. The unreacted amino groups on the resin were then reacted with methanol using the mixture of DCM, MeOH, and DIPEA (15:2:1) and the Fmoc-group of the AcA-grafted resin was then removed by 20% piperidine in NMP. After washing, the resin was mixed with Fmoc-O-(benzylphospho)-serine-OH (1 mmol) and DIPEA (4 mmol) and reacted in DCM (20 mL) for 4 h. After washing, the serine-grafted resin was treated with piperidine (20% in NMP). The following amino acid additions were performed using a tBu-protected Fmoc-Asp(O tBu)-OH/BOP/DIPEA (1.5:1.5:1.5, mmol) in NMP. Likewise, Fmoc-Gly-OH and Pbf-protected FMOC-Arg(Pbf)-OH were sequentially grafted on the resin to obtain a Fmoc-RGDS-resin. Then, five glycine sequences were grafted as a spacer to get Fmoc-G5-RGDS-resin. To introduce DOPA amino acid, acetonide-protected Fmoc-DOPA(acetonide)-OH (1 mmol) was used. A catechol-containing (DOPA)4 sequence was then grafted on the resin to obtain Fmoc-(DOPA)4-G5-RGDS-resin. After deprotection of Fmoc in the final amino acid, the amino groups were labeled with FITC. Then, the peptide-grafted resin was then incubated in 2% trifluoroacetic acid in DCM solution to cleave the peptide. After HPLC purification, we finally obtained a musselderived cancer-targeting peptide PEP-RGD (FITC-(DOPA)4-G5RGDS, yield: 20.3%). The synthesis of the control peptide PEP-RGE (FITC-(DOPA)4-G5-RGES) is similar as given above (yield: 19.2%). The two mussel-derived peptides were then characterized with ESIMS. FITC-(DOPA)4-G5-RGDS: ESI-MS (1937.9), 969.8 [M + 2H]2+; HPLC: 95.8% in purity. FITC-(DOPA)4-G5-RGES: ESI-MS (1951.93), 1952.6 [M + H]+, 977.0 [M + 2H]2+; HPLC: 96.3% in purity. Preparation of Prodrug Nanoparticles. The PEP-RGD or PEP-RGE (1.94 mg, 1 μmol) and BTZ (1.54 mg, 4 μmol) into 0.2 mL of dimethylsulfoxide (DMSO) were dissolved and stirred for 3 h to obtain the peptide-BTZ prodrug. PBS (2 mL, pH 7.4) was then added to the prodrug solution and sonicated for 5 min to obtain the BTZPEP-RGD or BTZ-PEP-RGE prodrug nanoparticles (yields around 88%). The morphology and size of the nanoparticles were examined by a transmission electron microscopy (TEM, Tecnai G2 20, FEI Company, Hillsboro, Oregon). The prodrug nanoparticles were centrifuged and lyophilized to determine the BTZ-loading efficiency using HPLC with a UV detector (LC 1200, Agilent Technologies, Santa Clara, CA), the wavelength for BTZ detection was 260 nm. Drug Release Properties. To examine the pH-responsiveness of BTZ release, dialysis bag diffusion technique was utilized. In brief, 1 mg of nanoparticles was dispersed in 2 mL of buffer solutions with different pH values and transferred to a dialysis bag (MWCO: 2000 Da). The sealed dialysis bag was then immersed in 15 mL of PBS media of different pH values. The incubation temperature was 37 °C. At preset time intervals, the amounts of released BTZ in the buffer solutions were determined by HPLC (100 μL). Cell Culture. Human embryonic kidney 293 cells (HEK 293) and MDA-MB-231 cells (human breast cancer cells) were cultured in (DMEM) supplemented with 10% FBS and 1% penicillin/ streptomycin in a humidified 5% CO2 atmosphere at 37 °C. In Vitro Cellular Uptake. The uptake of nanoparticles against the HEK 293 or MDA-MB-231 cells were performed using confocal laser scanning microscopy (CLSM) and flow cytometry. The cells (1 × 105) were maintained in DMEM supplemented with 10% FBS for 24 h on a 6-well culture plates. The medium was then changed by freshly prepared DMEM containing nanoparticles (50 μg/mL) for 4 h incubation. The unbound nanoparticles were then washed, and the distribution of the nanoparticles on the cells was characterized by CLSM. After that, trypsin was added to digest the nanoparticle-bound cells for 2 min, and the detached cells were collected by centrifugation. The fluorescent intensity was analyzed by a flow cytometer (BD Biosciences, San Jose, CA). The excitation and emission wavelengths were 490 and 525 nm, respectively. About 2 × 104 cells were collected, amplified, and scaled to obtain a singleparameter histogram.

pharmacokinetics of the BTZ-PEP-RGD and BTZ-PEP-RGE nanoparticles. In particular, the BTZ-PEP-RGD nanoparticles showed the best cancer inhibition in vivo, indicating the importance of tumor-specific uptake and rapid drug release in our peptide-based prodrug nanosystem. We further found that the peptide-based prodrug nanoparticles did not show any apparent side effects during the treatment. Although all of the tumor tissues in the groups of free BTZ, BTZ-PEP-RGD, and BTZ-PEP-RGE nanoparticles appeared in significantly large areas of necrosis or apoptosis, only the free BTZ-treated mice showed a retarded increase of body weight during the treatment (Figure 6c,d). These results ultimately verified the high cancer inhibition efficacy and the low systematic toxicity of our peptide-based prodrug nanosystem.

■

CONCLUSIONS We demonstrated a novel peptide-based prodrug nanocarrier for the pH-sensitive and cancer-targeting delivery of the antitumor drug BTZ in vitro and in vivo. The molecular structure of our peptide was inspired by the mussel-secreted proteins. It was designed with tumor-targeting RGD motifs and a fluorescent molecule FITC for imaging as well as tetravalent catechol groups for the dynamic conjugation of the BTZ via pH-sensitive boronic acid−catechol esters. Our studies found that the cancer-targeting ability, pH-responsive BTZ release, and the biogenicity of the mussel-derived peptide endowed the peptide-based prodrug nanocarrier with improved therapeutic efficiency and a relatively lower systematic toxicity both in vitro and in vivo. We believe the highly biomimetic nature of this peptide-based nanocarriers, the rapid drug release from the dynamically conjugated prodrugs, and the convenience of introducing cancer-targeting activity demonstrated in this work would provide new ideas for the development of biocompatible and biogenic drug delivery systems with improved chemotherapy efficacy in clinic.

■

EXPERIMENTAL SECTION

Materials. The acetonide-protected Fmoc-DOPA(acetonide)-OH was purchased from Okeanos Tech Co. Ltd. (Beijing, China). 2Chlorotritylchloride polymer resin was purchased from Alfa Aesar (Shanghai, China). The HPLC-grade solvent, fluorescein isothiocyanate (FITC), and 3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Shanghai, China). The Fmoc-protected amino acids, acetic anhydride (AcO2), N,N-diisopropylethylamine (DIPEA), and N-methyl-2-pyrrolidone (NMP) were purchased from J&K Scientific Ltd. (Shanghai, China) and used as received. Bortezomib (BTZ) was purchased from Dalian Meilun Biotech Co., Ltd (Dalian, China). Dichloromethane (DCM), triethylamine, Fmoc-6-aminocaproic acid (AcA), tetrahydrofuran, and N,N-dimethylformamide were dried with CaH2 and distilled before use. Phosphate buffer saline solution (PBS, 0.02 mmol/L, pH = 7.2) was prepared use MiniQ water (18.2 MΩ cm) and a purchased phosphate buffer salt (Beyotime Biotechnology, China). Trypsin/ ethylenediaminetetraacetic acid solution (0.25%), streptomycin, and penicillin were purchased from Gibco BRL. Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone. Live/Dead cell-staining Kit and Dead Cell Apoptosis Kit with annexin V-FITC, propidium iodide (PI), FITC-phalloidin, and 4′-6-diamidino-2-phenylindole were purchased from Enzo Life Sciences, Inc. and Invitrogen and were used as received. Peptide Synthesis. The mussel-derived peptides were prepared on 2-chlorotrityl chloride polymer resin using a standard Fmoc-based solid-phase synthesis strategy.47 The typical preparation procedure of FITC-(DOPA)4-G5-RGDS is presented as follow: 2-chlorotrityl 23954

DOI: 10.1021/acsami.9b09031 ACS Appl. Mater. Interfaces 2019, 11, 23948−23956

Research Article

ACS Applied Materials & Interfaces In Vitro Cytotoxicity. Cell viability after incubation with free BTZ, peptides, and the prodrug nanoparticles were examined by using MTT assay. Briefly, the HEK 293 or MDA-MB-231 cells were seeded in 96-well plates with a cell density of 1 × 104 per well and then incubated overnight. Then, the cells were incubated with different concentrations of nanoparticles at equivalent BTZ concentrations of 0, 10, 20, 50, and 100 nM. After 24 h, the media were replaced with DMEM containing MTT (5 mg/mL). After incubation for another 4 h, MTT was aspirated off and the formazan crystals were dissolved by adding DMSO. The absorbance was measured by a microplate reader at the wavelength of 570 nm. Live/Dead Cell Staining. For the observation on CLSM, the cells were seeded onto a confocal microscopy dish at a cell density of 1 × 104 per well. Prodrug nanoparticles with equivalent BTZ concentrations of 100 nM were added into the media and incubated for 24 h. Before imaging under CLSM, the cells were washed with PBS and then stained by using the Live/Dead cell-staining Kit according to the manufacturer’s protocol. Flow Cytometry Characterization of Cell Apoptosis. Cell apoptosis was examined using the Annexin V-FITC/PI Apoptosis Detection Kit. Briefly, the cells were seeded onto a 6-well dish at a density of 1 × 104 cells/well. Prodrug nanoparticles with a free BTZ at equivalent drug concentrations of 100 nM were added and incubated with the cells for 24 h. All of the cells were then collected and stained with the Annexin V-FITC/PI Apoptosis Detection Kit, followed by immediate flow cytometry analysis. Xenograft Tumor Model. The Institutional Animal Care and Use Committee of Sun Yat-sen University has approved all of the experiments for the human cancer cell lines and anima. Severe combined immune deficient mice were purchased from Guangdong Medical Laboratory Animal Center. MDA-MB-231 cells (2 × 106 cells/mouse/100 μL) in the DMEM medium were inoculated subcutaneously to the mice at the back. Tumors were measured by a vernier caliper and the volume (V) was calculated as 4π/3 × (length/2) × (width/2)2. In Vivo Pharmacokinetics. Normal Balb/c mice were intravenously injected with 200 μL of free BTZ or prodrug nanoparticles through the tail vein. At preset time intervals, 20 μL of the mouse blood was drawn and then dissolved in 300 μL of lysis buffer.52 Three hundred microliters of HCl/isopropanol was added to extract BTZ from blood. The mixture was then incubated in dark overnight and centrifuged to obtain the BTZ supernatant. The amounts of BTZ in blood system was then quantified by HPLC. In Vivo Cancer Inhibition. The antitumor treatments were performed when the size of tumors on the back of the cancer xenograft mice reached 100 mm3. The tumor-bearing mice (n = 5 for each group) were intravenously injected with saline, PEP-RGD, BTZ drug, BTZ-PEP-RGE, and BTZ-PEP-RGD prodrug nanoparticles in the tail veins. The injection was fixed at a single dose of 2 mg BTZ/kg in PBS every 2 days. The changes of body weights and tumor sizes on the mice were then recorded every 2 days. After 20 days of treatment, all of the mice were sacrificed and the tumor tissues were collected for further staining. Immunohistochemical Analysis. The collected tumor tissues were fixed with paraformaldehyde and then embedded in paraffin. The paraffin-embedded tissues were then sliced into 4 μm thickness, and hematoxylin eosin (H&E) staining was then carried out to evaluate the apoptosis of tumor tissues. Statistical Analysis. All of the data were presented as mean ± standard deviation. The Student’s t-test was performed via SPSS 16.0 software, and the value of probability (p) was used to indicate statistical significance (*p < 0.01 and **p < 0.001).

■

Guoqing Pan: 0000-0001-5187-796X Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We greatly acknowledge the financial support from the National Natural Science Foundation of China (21875092, 21574091, 91649204, 21706099, and 51801076), the Natural Science Foundation of Jiangsu Province (BK20160056 and BK20160491), and the “Six Talent Peaks” program of Jiangsu Province (2018-XCL-013).

■

REFERENCES

(1) Chabner, B. A.; Roberts, T. G. Chemotherapy and the War on Cancer. Nat. Rev. Cancer 2005, 5, 65−72. (2) Shapiro, C. L.; Recht, A. Side Effects of Adjuvant Treatment of Breast Cancer. N. Engl. J. Med. 2001, 344, 1997−2008. (3) Shi, J.; Kantoff, P. W.; Wooster, R.; Farokhzad, O. C. Cancer nanomedicine: progress, challenges and opportunities. Nat. Rev. Cancer 2017, 17, 20. (4) Markman, J. L.; Rekechenetskiy, A.; Holler, E.; Ljubimova, J. Y. Nanomedicine Therapeutic Approaches to Overcome Cancer Drug Resistance. Adv. Drug Delivery Rev. 2013, 65, 1866−1879. (5) Park, J. H.; Lee, S.; Kim, J.-H.; Park, K.; Kim, K.; Kwon, I. C. Polymeric Nanomedicine for Cancer Therapy. Prog. Polym. Sci. 2008, 33, 113−137. (6) Wang, J.; Sun, X.; Mao, W.; Sun, W.; Tang, J.; Sui, M.; Shen, Y.; Gu, Z. Tumor Redox Heterogeneity-Responsive Prodrug Nanocapsules for Cancer Chemotherapy. Adv. Mater. 2013, 25, 3670− 3676. (7) Chen, Q.; Liu, G.; Liu, S.; Su, H.; Wang, Y.; Li, J.; Luo, C. Remodeling the Tumor Microenvironment with Emerging Nanotherapeutics. Trends Pharmacol. Sci. 2018, 39, 59−74. (8) Li, N.; Zhao, L.; Qi, L.; Li, Z.; Luan, Y. Polymer assembly: Promising Carriers as Co-delivery Systems for Cancer Therapy. Prog. Polym. Sci. 2016, 58, 1−26. (9) Liong, M.; Lu, J.; Kovochich, M.; Xia, T.; Ruehm, S. G.; Nel, A. E.; Tamanoi, F.; Zink, J. I. Multifunctional Inorganic Nanoparticles for Imaging, Targeting, and Drug Delivery. ACS Nano 2008, 2, 889− 896. (10) Huang, H.-C.; Barua, S.; Sharma, G.; Dey, S. K.; Rege, K. Inorganic Nanoparticles for Cancer Imaging and Therapy. J. Controlled Release 2011, 155, 344−357. (11) Cui, D.; Huang, J.; Zhen, X.; Li, J.; Jiang, Y.; Pu, K. A Semiconducting Polymer Nano-prodrug for Hypoxia-Activated Photodynamic Cancer Therapy. Angew. Chem., Int. Ed. 2019, 58, 5920−5924. (12) Li, J.; Xie, C.; Huang, J.; Jiang, Y.; Miao, Q.; Pu, K. Semiconducting Polymer Nanoenzymes with Photothermic Activity for Enhanced Cancer Therapy. Angew. Chem., Int. Ed. 2018, 57, 3995−3998. (13) Gunawan, B.; Kaplowitz, N. Clinical Perspectives on Xenobiotic-Induced Hepatotoxicity. Drug Metab. Rev. 2004, 36, 301−312. (14) Pan, G.; Liu, S.; Zhao, X.; Zhao, J.; Fan, C.; Cui, W. FullCourse Inhibition of Biodegradation-Induced Inflammation in Fibrous Scaffold by Loading Enzyme-Sensitive Prodrug. Biomaterials 2015, 53, 202−210. (15) Ceonzo, K.; Gaynor, A.; Shaffer, L.; Kojima, K.; Vacanti, C. A.; Stahl, G. L. Polyglycolic Acid-Induced Inflammation: Role of Hydrolysis and Resulting Complement Activation. Tissue Eng. 2006, 12, 301−308. (16) Hotamisligil, G. S. Inflammation and Metabolic Disorders. Nature 2006, 444, 860−867. (17) Testa, B. Prodrug Research: Futile or Fertile? Biochem. Pharmacol. 2004, 68, 2097−2106.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yue Ma: 0000-0002-1849-1384 Xiaowei Zeng: 0000-0002-2804-2689 23955

DOI: 10.1021/acsami.9b09031 ACS Appl. Mater. Interfaces 2019, 11, 23948−23956

Research Article

ACS Applied Materials & Interfaces (18) Kratz, F.; Müller, I. A.; Ryppa, C.; Warnecke, A. Prodrug Strategies in Anticancer Chemotherapy. ChemMedChem 2008, 3, 20− 53. (19) Nicolas, J. Drug-Initiated Synthesis of Polymer Prodrugs: Combining Simplicity and Efficacy in Drug Delivery. Chem. Mater. 2016, 28, 1591−1606. (20) Rautio, J.; Meanwell, N. A.; Di, L.; Hageman, M. J. The Expanding Role of Prodrugs in Contemporary Drug Design and Development. Nat. Rev. Drug Discovery 2018, 17, 559−587. (21) Cheetham, A. G.; Chakroun, R. W.; Ma, W.; Cui, H. SelfAssembling Prodrugs. Chem. Soc. Rev. 2017, 46, 6638−6663. (22) Hu, X.; Chai, Z.; Lu, L.; Ruan, H.; Wang, R.; Zhan, C.; Xie, C.; Pan, J.; Liu, M.; Wang, H.; Lu, W. Bortezomib Dendrimer ProdrugBased Nanoparticle System. Adv. Funct. Mater. 2019, 29, No. 1807941. (23) Su, J.; Chen, F.; Cryns, V. L.; Messersmith, P. B. Catechol Polymers for pH-Responsive, Targeted Drug Delivery to Cancer Cells. J. Am. Chem. Soc. 2011, 133, 11850−11853. (24) Daum, S.; Reshetnikov, M. S. V.; Sisa, M.; Dumych, T.; Lootsik, M. D.; Bilyy, R.; Bila, E.; Janko, C.; Alexiou, C.; Herrmann, M.; Sellner, L.; Mokhir, A. Lysosome-Targeting Amplifiers of Reactive Oxygen Species as Anticancer Prodrugs. Angew. Chem., Int. Ed. 2017, 56, 15545−15549. (25) Liu, J.; Liu, W.; Weitzhandler, I.; Bhattacharyya, J.; Li, X.; Wang, J.; Qi, Y.; Bhattacharjee, S.; Chilkoti, A. Ring-Opening Polymerization of Prodrugs: A Versatile Approach to Prepare WellDefined Drug-Loaded Nanoparticles. Angew. Chem., Int. Ed. 2015, 54, 1002−1006. (26) Zheng, Y.; Yu, B.; Ji, K.; Pan, Z.; Chittavong, V.; Wang, B. Esterase-Sensitive Prodrugs with Tunable Release Rates and Direct Generation of Hydrogen Sulfide. Angew. Chem., Int. Ed. 2016, 55, 4514−4518. (27) Russo, A.; Bronte, G.; Fulfaro, F.; Cicero, G.; Adamo, V.; Gebbia, N.; Rizzo, S. Bortezomib: A New Pro-Apoptotic Agent in Cancer Treatment. Curr. Cancer Drug Targets 2010, 10, 55−67. (28) Kane, R. C.; Farrell, A. T.; Sridhara, R.; Pazdur, R. United States Food and Drug Administration Approval Summary: Bortezomib for the Treatment of Progressive Multiple Myeloma after One Prior Therapy. Clin. Cancer Res. 2006, 12, 2955−2960. (29) Choe, U.-J.; Sun, V. Z.; Tan, J.-K. Y.; Kamei, D. T. SelfAssembled Polypeptide and Polypeptide Hybrid Vesicles: From Synthesis to Application. In Peptide-Based Materials; Deming, T., Ed.; Springer: Berlin, 2012; pp 117−134. (30) Deming, T. J. Synthetic Polypeptides for Biomedical Applications. Prog. Polym. Sci. 2007, 32, 858−875. (31) Wu, Y.; MacKay, J. A.; McDaniel, J. R.; Chilkoti, A.; Clark, R. L. Fabrication of Elastin-Like Polypeptide Nanoparticles for Drug Delivery by Electrospraying. Biomacromolecules 2009, 10, 19−24. (32) Andrew MacKay, J.; Chen, M.; McDaniel, J. R.; Liu, W.; Simnick, A. J.; Chilkoti, A. Self-Assembling Chimeric Polypeptide− Doxorubicin Conjugate Nanoparticles that Abolish Tumours after a Single Injection. Nat. Mater. 2009, 8, 993. (33) Peng, M.; Qin, S.; Jia, H.; Zheng, D.; Rong, L.; Zhang, X. Selfdelivery of a Peptide-Based Prodrug for Tumor-Targeting Therapy. Nano Res. 2016, 9, 663−673. (34) Zhang, X.-X.; Eden, H. S.; Chen, X. Peptides in Cancer Nanomedicine: Drug Carriers, Targeting Ligands and Protease Substrates. J. Controlled Release 2012, 159, 2−13. (35) Vivès, E.; Schmidt, J.; Pèlegrin, A. Cell-Penetrating and CellTargeting Peptides in Drug Delivery. Biochim. Biophys. Acta 2008, 1786, 126−138. (36) Wu, X. L.; Kim, J. H.; Koo, H.; Bae, S. M.; Shin, H.; Kim, M. S.; Lee, B.-H.; Park, R.-W.; Kim, I.-S.; Choi, K.; Kwon, I. C.; Kim, K.; Lee, D. S. Tumor-Targeting Peptide Conjugated pH-Responsive Micelles as a Potential Drug Carrier for Cancer Therapy. Bioconjugate Chem. 2010, 21, 208−213. (37) Bhutia, S. K.; Maiti, T. K. Targeting Tumors with Peptides from Natural Sources. Trends Biotechnol. 2008, 26, 210−217.

(38) Laakkonen, P.; Porkka, K.; Hoffman, J. A.; Ruoslahti, E. A Tumor-Homing Peptide with a Targeting Specificity Related to Lymphatic Vessels. Nat. Med. 2002, 8, 751−755. (39) Danhier, F.; Le Breton, A.; Préat, V. RGD-Based Strategies To Target Alpha(v) Beta(3) Integrin in Cancer Therapy and Diagnosis. Mol. Pharmaceutics 2012, 9, 2961−2973. (40) Wei, Q.; Achazi, K.; Liebe, H.; Schulz, A.; Noeske, P. L. M.; Grunwald, I.; Haag, R. Mussel-Inspired Dendritic Polymers as Universal Multifunctional Coatings. Angew. Chem., Int. Ed. 2014, 53, 11650−11655. (41) Lee, H.; Scherer, N. F.; Messersmith, P. B. Single-Molecule Mechanics of Mussel Adhesion. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 12999−13003. (42) Dalsin, J. L.; Hu, B.-H.; Lee, B. P.; Messersmith, P. B. Mussel Adhesive Protein Mimetic Polymers for the Preparation of Nonfouling Surfaces. J. Am. Chem. Soc. 2003, 125, 4253−4258. (43) Narkar, A. R.; Barker, B.; Clisch, M.; Jiang, J.; Lee, B. P. pH Responsive and Oxidation Resistant Wet Adhesive based on Reversible Catechol−Boronate Complexation. Chem. Mater. 2016, 28, 5432−5439. (44) Liu, L.; Tian, X.; Ma, Y.; Duan, Y.; Zhao, X.; Pan, G. A Versatile Dynamic Mussel-Inspired Biointerface: From Specific Cell Behavior Modulation to Selective Cell Isolation. Angew. Chem., Int. Ed. 2018, 57, 7878−7882. (45) Liu, S.; Pan, J.; Liu, J.; Ma, Y.; Qiu, F.; Mei, L.; Zeng, X.; Pan, G. Dynamically PEGylated and Borate-Coordination-Polymer-Coated Polydopamine Nanoparticles for Synergetic Tumor-Targeted, Chemo-Photothermal Combination Therapy. Small 2018, 14, No. 1703968. (46) Wang, C.; Zhao, T.; Li, Y.; Huang, G.; White, M. A.; Gao, J. Investigation of Endosome and Lysosome Biology by Ultra pHSensitive Nanoprobes. Adv. Drug Deliverery Rev. 2017, 113, 87−96. (47) Pan, G.; Sun, S.; Zhang, W.; Zhao, R.; Cui, W.; He, F.; Huang, L.; Lee, S.-H.; Shea, K. J.; Shi, Q.; Yang, H. Biomimetic Design of Mussel-Derived Bioactive Peptides for Dual-Functionalization of Titanium-Based Biomaterials. J. Am. Chem. Soc. 2016, 138, 15078− 15086. (48) Zhao, H.; Huang, Y.; Zhang, W.; Guo, Q.; Cui, W.; Sun, Z.; Eglin, D.; Liu, L.; Pan, G.; Shi, Q. Mussel-Inspired Peptide Coatings on Titanium Implant to Improve Osseointegration in Osteoporotic Condition. ACS Biomater. Sci. Eng. 2018, 4, 2505−2515. (49) Pan, G.; Guo, B.; Ma, Y.; Cui, W.; He, F.; Li, B.; Yang, H.; Shea, K. J. Dynamic Introduction of Cell Adhesive Factor via Reversible Multicovalent Phenylboronic Acid/cis-Diol Polymeric Complexes. J. Am. Chem. Soc. 2014, 136, 6203−6206. (50) Pan, G.; Shinde, S.; Yeung, S. Y.; Jakštaitė, M.; Li, Q.; Wingren, A. G.; Sellergren, B. An Epitope-Imprinted Biointerface with Dynamic Bioactivity for Modulating Cell−Biomaterial Interactions. Angew. Chem., Int. Ed. 2017, 56, 15959−15963. (51) Pan, G.; Guo, Q.; Ma, Y.; Yang, H.; Li, B. Thermo-Responsive Hydrogel Layers Imprinted with RGDS Peptide: A System for Harvesting Cell Sheets. Angew. Chem., Int. Ed. 2013, 52, 6907−6911. (52) Zeng, X.; Liu, G.; Tao, W.; Ma, Y.; Zhang, X.; He, F.; Pan, J.; Mei, L.; Pan, G. A Drug-Self-Gated Mesoporous Antitumor Nanoplatform Based on pH-Sensitive Dynamic Covalent Bond. Adv. Funct. Mater. 2017, 27, No. 1605985. (53) Zhang, L.; Zhang, X.; Lu, G.; Li, F.; Bao, W.; Song, C.; Wei, W.; Ma, G. Cell Membrane Camouflaged Hydrophobic Drug Nanoflake Sandwiched with Photosensitizer for Orchestration of ChemoPhotothermal Combination Therapy. Small 2019, 15, No. 1805544.

23956

DOI: 10.1021/acsami.9b09031 ACS Appl. Mater. Interfaces 2019, 11, 23948−23956