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Yinuo Li, Yoshitaka Matsumoto, [Lili Chen](https://orcid.org/0000-0001-8290-5230), Yu Sugawara, [Emiho Oe](https://orcid.org/0000-0001-7326-7740), [Nanami Fujisawa](https://orcid.org/0000-0002-8894-1790), [Mitsuhiro Ebara](https://orcid.org/0000-0002-7906-0350), Hideyuki Sakurai

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[Smart Nanofiber Mesh with Locally Sustained Drug Release Enabled Synergistic Combination Therapy for Glioblastoma](https://mdr.nims.go.jp/datasets/a2e0fb18-037c-45bf-a122-dff44f138d35)

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Smart Nanofiber Mesh with Locally Sustained Drug Release Enabled Synergistic Combination Therapy for Glioblastoma5.719ArticleSmart Nanofiber Mesh with LocallySustained Drug Release EnabledSynergistic Combination Therapyfor GlioblastomaYinuo Li, Yoshitaka Matsumoto, Lili Chen, Yu Sugawara, Emiho Oe, Nanami Fujisawa,Mitsuhiro Ebara and Hideyuki SakuraiSpecial IssueBiohybrid Nanofibers and Nanomaterial-Contained Fibers: Fabrication and ApplicationEdited byDr. Shaohua Wu and Dr. Wenwen Zhaohttps://doi.org/10.3390/nano13030414https://www.mdpi.com/journal/nanomaterialshttps://www.ncbi.nlm.nih.gov/pubmed/?term=2079-4991https://www.mdpi.com/journal/nanomaterials/statshttps://www.mdpi.com/journal/nanomaterials/special_issues/Biohybrid_Nanofibershttps://www.mdpi.comhttps://doi.org/10.3390/nano13030414Citation: Li, Y.; Matsumoto, Y.; Chen,L.; Sugawara, Y.; Oe, E.; Fujisawa, N.;Ebara, M.; Sakurai, H. SmartNanofiber Mesh with LocallySustained Drug Release EnabledSynergistic Combination Therapy forGlioblastoma. Nanomaterials 2023, 13,414. https://doi.org/10.3390/nano13030414Academic Editor: Jose L. AriasReceived: 29 December 2022Revised: 16 January 2023Accepted: 17 January 2023Published: 19 January 2023Copyright: © 2023 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).nanomaterialsArticleSmart Nanofiber Mesh with Locally Sustained Drug ReleaseEnabled Synergistic Combination Therapy for GlioblastomaYinuo Li 1 , Yoshitaka Matsumoto 2,3,* , Lili Chen 4 , Yu Sugawara 3, Emiho Oe 4,5 , Nanami Fujisawa 4,5 ,Mitsuhiro Ebara 4 and Hideyuki Sakurai 2,31 Department of Radiation Oncology, Graduate School of Comprehensive Human Sciences, University ofTsukuba, Tsukuba 305-8575, Japan2 Department of Radiation Oncology, Clinical Medicine, Faculty of Medicine, University of Tsukuba,Tsukuba 305-8575, Japan3 Proton Medical Research Center, University of Tsukuba Hospital, Tsukuba 305-8576, Japan4 Research Center for Functional Materials, National Institute for Materials Science (NIMS), 1-1 Namiki,Tsukuba 305-0044, Japan5 Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba 305-0006, Japan* Correspondence: ymatsumoto@pmrc.tsukuba.ac.jp; Tel.: +81-29-853-7100; Fax: +81-29-853-7102Abstract: This study aims to propose a new treatment model for glioblastoma (GBM). The combina-tion of chemotherapy, molecular targeted therapy and radiotherapy has been achieved in a highlysimultaneous manner through the application of a safe, non-toxic, locally sustained drug-releasingcomposite Nanofiber mesh (NFM). The NFM consisted of biodegradable poly(ε-caprolactone) withtemozolomide (TMZ) and 17-allylamino-17-demethoxygeldanamycin (17AAG), which was usedin radiation treatment. TMZ and 17AAG combination showed a synergistic cytotoxicity effect inthe T98G cell model. TMZ and 17AAG induced a radiation-sensitization effect, respectively. TheNFM containing 17AAG or TMZ, known as 17AAG-NFM and TMZ-NFM, enabled cumulative drugrelease of 34.1% and 39.7% within 35 days. Moreover, 17AAG+TMZ-NFM containing both drugsrevealed a synergistic effect in relation to the NFM of a single agent. When combined with radiation,17AAG+TMZ-NFM induced in an extremely powerful cytotoxic effect. These results confirmed theapplication of NFM can simultaneously allow multiple treatments to T98G cells. Each modalityachieved a significant synergistic effect with the other, leading to a cascading amplification of thetherapeutic effect. Due to the superior advantage of sustained drug release over a long periodof time, NFM has the promise of clinically addressing the challenge of high recurrence of GBMpost-operatively.Keywords: glioblastoma; combination therapy; synergistic effect; TMZ; 17AAG; radiation therapy;radiosensitization; nanofiber1. IntroductionGlioblastoma (GBM) is the most common type of malignant brain tumor in adults,with a poor prognosis of a five-year overall survival rate of less than 10% [1]. Because ofthe diffuse and infiltrative nature of GBM, tumor cells often remain around the surgicalresection site, making recurrence possible [2]. Patients with GBM have a postoperativerecurrence rate of 80% to 90% [3], and there is no efficient treatment for recurrent GBM [4].In addition to surgery, a powerful combination of treatments is necessary to eradicate GBM,which includes killing the primary tumor and the cells that have invaded the surroundingtissue.Heat-shock protein 90 (Hsp90) is a molecular chaperone associated with the stabilityand function of some over-expressed signal proteins that promote the growth and survivalof cancer cells [5]. Hsp90 is shown to induce resistance to radiotherapy and chemotherapyNanomaterials 2023, 13, 414. https://doi.org/10.3390/nano13030414 https://www.mdpi.com/journal/nanomaterialshttps://doi.org/10.3390/nano13030414https://doi.org/10.3390/nano13030414https://creativecommons.org/https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://www.mdpi.com/journal/nanomaterialshttps://www.mdpi.comhttps://orcid.org/0000-0002-0423-2541https://orcid.org/0000-0002-2074-3042https://orcid.org/0000-0001-8290-5230https://orcid.org/0000-0001-7326-7740https://orcid.org/0000-0002-8894-1790https://orcid.org/0000-0002-7906-0350https://doi.org/10.3390/nano13030414https://www.mdpi.com/journal/nanomaterialshttps://www.mdpi.com/article/10.3390/nano13030414?type=check_update&version=1Nanomaterials 2023, 13, 414 2 of 13as well as contribute to local recurrence and distant metastasis [6]. The 17-allylamino-17-demethoxygeldanamycin (17AAG) is the first Hsp90 inhibitor to enter into clinical trials [7],which can bind and promote the degradation of Hsp90 client proteins [8]. Markedly, inGBM, Hsp90 client proteins are known to be disordered, including EGFR, PDGFR, Akt, andvariant P53 [9]. Therefore, the molecular targeted drug 17AAG is an excellent candidatefor multi-target therapy of GBM, which may provide a better prognosis. Furthermore,17AAG has been shown to enhance the radiosensitivity of tumor cell lines, while having noinfluence on that of normal human fibroblast cell lines [10]. Some studies have suggestedthat 17AAG may downregulate Rad51 and BRCA2 protein levels, which are related proteinsfor DNA repair after radiation-induced damage [11,12].Postoperative systemic temozolomide (TMZ) chemotherapy combined with radiationtherapy is the standard regimen for the clinical practice of GBM [13]. Oral chemothera-peutic TMZ, an alkylating agent, can cross the blood-brain barrier (BBB). In order to formthe active intermediate 5-(3-methyltriazen-1-yl) imidazole-4-carboxamide (MTIC), watermolecules undergo a nucleophilic reaction. This intermediate is then converted to 4-amino-5-imidazole-carboxamide (AIC), causing DNA methylation and DNA damage [14,15].Gastrointestinal irritation and vomiting were the main adverse effects in patients afterTMZ oral administration [16]. In addition, although TMZ can cross the BBB, its abilityto enter brain tissue remains limited, which makes it clinically necessary to increase theapplied concentration of the drug and further raises the risk of side effects [17,18]. Thus,the delivery system for local low-dose drug release in brain tissue is highly desired to bedeveloped to solve these problems.To improve drug aggregation and sustained release in local tissues over long dura-tions, various nanocarriers have been designed and synthesized based on nano-syntheticchemistry [19]. Particularly, poly-caprolactone (PCL) biomaterials have gained considerableattention due to their high biocompatibility, ease of implantability, and manipulation [20].This biomaterial has been approved by the U.S. Food and Drug Administration for a widerange of biomedical applications because of its safety and efficiency in human tissue engi-neering and cancer drug delivery [21]. The PCL- nanofiber mesh (NFM), which is madefrom PCL biomaterials, has already shown some results in treating breast cancer and lungcancer by combining chemotherapy, radiotherapy, and hyperthermia [22,23].Currently, electrospinning technology has been used in a wide range of fields for itsunique advantages. The synthesis of nanofibrous materials for biomedicine by electro-spinning has attracted considerable attention owing to their small diameter, high specificsurface area, appropriate porosity, and controlled shape [24]. Most importantly, theirnanofiber structure is highly similar to the structural features of the extracellular matrix(ECM) and can effectively promote cell adhesion, growth, migration, proliferation andeven ECM remodeling and new tissue regeneration. In addition, numerous studies havedemonstrated that electrospun nanofibers may have good antibacterial effects, which issignificant for the promotion of human health [25].This study aims to develop a new sustained-release drug-delivery system for localimplantation in GBM to optimize anticancer efficacy and reduce side effects and recurrencerates by providing simultaneous and consistent multimodal effects of radiation therapy,chemotherapy, and molecular targeted therapy. As illustrated in Scheme 1, the chemothera-peutic drug TMZ is integrated with 17AAG in a nano-platform NFM, which is made ofelectrospun PCL materials and implanted in local brain tissue lesions immediately aftertumor resection by surgery. Radiation therapy was administered while the drug wasconsistently released locally from the NFM.Nanomaterials 2023, 13, 414 3 of 13 ’TMZ (150 μM, 500 μM)Scheme 1. Smart nanofiber mesh (NFM) system with local sustained drug release is surgically im-planted to achieve the combined antitumor effect of radiation therapy, chemotherapy, and moleculartargeted therapy.2. Materials and MethodsCell culture and drugs. A human cell line derived from GBM multiforme T98G cells(RCB1954) was purchased from the RIKEN BioResource Research Center. Cells weremaintained in Eagle’s minimum essential medium (E-MEM; Sigma-Aldrich, Tokyo, Japan)supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillinand 100 mg/mL streptomycin; Sigma-Aldrich) in a 5% CO2 incubator at 37 ◦C for furtherexperiments. The 17AAG was obtained from Sigma-Aldrich Japan (Tokyo, Japan). TMZwas purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).Colony formation assay. Cell survival curves were determined by colony formationassay. Cells were seeded in 6 cm dishes or T25 flasks, incubated in a 5% CO2 incubatorat 37 ◦C for 48 h, and treated with drugs or irradiated with X-rays. After drug treatmentor irradiation, cells were washed with PBS, separated from the dishes by 0.02% trypsinprocessing, diluted with a fresh medium, counted, and diluted. Cell suspensions expectedto produce approximately 100 surviving cells were seeded into six cm culture dishes intriplicate and cultured for 14 days. Subsequently, the cells were fixed and stained with 10%formalin solution and 1% methylene blue solution (20% MtOH). The number of colonieswas counted, with colonies consisting of 50 or more as significant colonies, and this wasplotted as the cell survival rate to produce a cell survival curve.Sensitivity and efficacy of drug combinations. Approximately 1.5 × 105/3 mL ofT98G cell solutions were added to six-well plates and incubated for 48 h. Subsequently,the medium was changed with the prepared medium containing different concentrationsof TMZ (150 µM, 500 µM), 17AAG (100 nM, 200 nM, 350 nM), or TMZ-17AAG solutionmixture, and continually the six-well plates were placed in an incubator at 37 ◦C and 5%CO2 for 24 h. Finally, the number of colonies was counted by means of a colony formationassay to calculate the cell survival rate in the case of different drug treatments. Each of theabove experiments was performed at least three times. The drug combination efficacy wasassessed by application of the combination index (CI) calculation [26]. The formula was asfollows:Combination index (CI) = D1/DX1 + D2/DX2, (1)Nanomaterials 2023, 13, 414 4 of 13where D1 is the half maximal inhibitory concentration (IC50) of 17AAG under the17AAG+TMZ combination circumstance; DX1 is the IC50 when 17AAG is administeredalone; and D2 is the IC50 of TMZ under a two-drug combination, and DX2 is the IC50 whenTMZ is applied alone. It is well known that when the CI < 1, it means that TMZ and 17AAGhave a synergistic effect in the T98G cell model; when CI = 1, it represents an additiveeffect; and when CI > 1, it indicates an antagonistic effect between TMZ and 17AAG duringcombination administration.Irradiation and radiosensitization of drugs. T98G cell suspensions containing2.5 × 105/5 mL were added to T25 flasks and incubated for 48 h. Cultured T98G cellswere treated with different concentrations of 17AAG (100 nM, 200 nM) or TMZ (150 µM,500 µM) solutions for 24 h. Irradiation time was adjusted so that samples containing eachconcentration of 17AAG were irradiated at 0.8, 1.5, 2, 4, and 6 Gy, respectively. Samplescontaining TMZ were irradiated at doses of 0.8, 1.5, 3, and 6 Gy. A colony formation assaywas conducted immediately after irradiation, and survival curves were obtained basedon the survival rate with different irradiation and drug combinations. In this experiment,the sensitizing effect of TMZ and 17AAG drugs on radiation was determined by the sensi-tizer enhancement ratios (SERs). The SER is the ratio of the irradiation dose required toachieve a specific biological effect when irradiated alone and the irradiation dose requiredto achieve the same biological effect when irradiation is combined with the application of aradiosensitizer (such as drugs) [27]; after the application of a radiosensitizer, the irradiationdose can be reduced to achieve a specific biological effect when irradiated alone. SER > 1indicates that the drug has a radiosensitization effect, and higher values suggest a moresubstantial sensitization effect.SER = D0/DC (for a certain survival) (2)D0 is the radiation dose required for a certain survival rate (50% in this study) whenirradiated alone. DC is the radiation dose needed for the same survival rate when the drugsare combined.Fabrication and characterization of nanofiber mesh. Poly(ε-caprolactone) (PCL,Mw = 80 kDa) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).Synthesis of PCL-NFM utilized electrospinning technology (Nanon-01A, MECC Co., Ltd.,Japan). The PCL-NFM was made according to a previously published protocol [28]. The17AAG and TMZ were dissolved in HFIP solution at a concentration of 1.72 % (w/v) and0.007% (w/v), respectively, then the PCL solution was added prior to electrospinning, andthe mixture was well stirred. A positive voltage of 20 kV was applied to the solution toallow polymer injection to form. The flow rate was set at 1.0 mL/h, the separation ofthe needle at 13 cm (25 gauge), and the collector plate at 25 ◦C and 42% humidity. Thenanofibers were then collected on a plate 8 cm from the syringe needle. The morphology ofPCL-NFM was investigated through a scanning electron microscopy (SEM, SU8000, HitachiHigh-Technologies Corporation, Tokyo, Japan). The micrographs were recorded at 15 kVwith a magnification of 1000×. The diameters of nanofibers were measured using image J.For the mechanical analysis properties of the PCL-NFM, the ultimate tensile strength(UTS) was determined by a tensile tester (EZ-S 500N, Shimadzu, Kyoto, Japan). Allexperiments were conducted at an elongation rate of 5 mm/min under room temperature(n = 3). The sample thickness was 0.5 mm, and the width was 50 mm. The tests were carriedout until the sample broke.Furthermore, the thermal properties of the PCL-NFM were characterized using differ-ential scanning calorimetry (DSC) (7000X, Hitachi High-Tech Science, Japan). All sampleswere first equilibrated at 200 ◦C and cooled to 0 ◦C. The DSC curves were obtained in thesecond heating run at a rate of 15 ◦C min−1 for all runs.Long-term drug release behavior. After UV-Vis spectrum analysis of 17AAG andTMZ, the drug absorbance-wavelength standard curves of 17AAG and TMZ were detected,respectively. Each piece of the NFM (40 mg in weight) containing 1.72 % of TMZ or 0.007%of 17AAG was submerged in 5 mL of PBS solution in a glass vial and maintained at 37 ◦CNanomaterials 2023, 13, 414 5 of 13for 35 days. At certain time intervals, 3 mL of PBS-containing drugs was withdrawn, andthe same volume of fresh PBS was added. According to the wavelengths detected above,using a plate reader (Infinite 200PRO, Tecan, Männedorf, Switzerland), the fluorescenceand absorbance of TMZ and 17AAG could be measured separately. Under PBS conditions,the cumulative drug release (%) of TMZ and 17AAG over 35 days could be calculated.Cytotoxic effects with nanofiber mesh in vitro. The antitumor effect of the NFM wasexamined with human GBM multiforme T98G cells. T98G cell solution with a targetconcentration of approximately 2.5 × 105/5 mL was added to the T25 flasks and incubatedfor 48 h. Subsequently, the medium was removed, a piece of NFM (weight 40 mg, containing1.72% TMZ or 0.007% 17AAG) was applied, and 5 mL of fresh medium was added again aswell as kept in an incubator at 37 ◦C and 5% CO2 for 24 h. After 24 h, the irradiation wascarried out with an X-ray device (130 kV, 0.57 Gy/min) as described above for 158 s at anexposure dose of 1.5 Gy. Following the irradiation, cell survival and the antitumor effectunder NFM administration were calculated by colony formation assay.Statistical analysis. All the data are presented as means ± standard deviation (SD).Statistical analysis was performed using Student’s t-test and one-way analysis of variance(ANOVA) using GraphPad Prism software version 9.0 (GraphPad Software, San Diego, CA,USA). The p-value less than 0.05 (p < 0.05) was considered statistically significant betweenthe results.3. Results3.1. Drug Sensitivity ResultsThe effects of 17AAG and TMZ on T98G cells were investigated individually for 24 h oftreatment. Both agents impacted and decreased cell survival in a dose-dependent manner,as shown in Figure 1. The concentration ranges of 15–960 nM of 17AAG in Figure 1a and100–800 µM of TMZ in Figure 1b were examined on T98G cells, and the IC50 values weredetermined. It was found that 17AAG and TMZ had IC50 concentrations of 376.4 nM and659.7 µM for 24 h treatment of T98G cells, respectively (p < 0.01).’–– (a) (b) Figure 1. Drug sensitivity results of 17AAG and TMZ. (a) Survival curve for T98G cells with 24 htreatment of 17AAG; (b) Survival curve for T98G cells with 24 h treatment of TMZ.3.2. Synergistic Effects between TMZ and 17AAGTo determine the drug interaction between 17AAG and TMZ, 17AAG at concentrationsof 100, 200, and 350 nM was administered in combination with TMZ at 150 µM and 500 µMto T98G cells treated for 24 h. The cell survival curves under the drug combination(Figure 2) were obtained according to the colony formation assay. The IC50 concentrationsof 17AAG and TMZ under the combination of two drugs were decreased to 148.2 nM(D1) and 156.9µM (D2), respectively. From the equation in (1), as mentioned above, theCI = 0.632 < 1 means that the combination of 17AAG and TMZ, when applied to T98G,exhibited a significant drug synergistic effect. Although we did not measure the survivalrate at the specific concentration of 376.4 nM (the IC50 concentration of 17AAG in Figure 1),Nanomaterials 2023, 13, 414 6 of 13it can be estimated that the IC50 concentration of 17AAG in Figure 2 became over 400 nMwhich is much bigger than 376.4 nM. We consider that this may be due to the slight variationbetween the actual and expected concentrations caused by human manipulation duringthe process of the dilutions of 17AAG. Because the units of IC50 for 17AAG are very smallat the nanomolar scale, they would be very susceptible to errors.  Figure 2. Surviving fraction of 24 h treatment of TMZ combined with 17AAG for T98G cells.3.3. Radiosensitization of 17AAG and TMZIn this study, the radiation-sensitizing effects of 17AAG and TMZ were tested in theGBM T98G model, respectively. Cells treated with 100 nM and 200 nM of 17AAG wereirradiated with different X-rays in Figure 3a. Cells treated with 150 µM and 500 µM of TMZwere also irradiated with different X-ray doses, shown in Figure 3b. The SERs evaluatedthe radiosensitization, which is calculated by the radiation dose required when treatingwith radiation alone and when combined with drugs to achieve a specific survival rate.According to equation (2), the SERs were 1.30 and 1.56 for treatment with 100 nM and200 nM 17AAG, at 50% survival, and 1.18 and 1.67 for treatment with 150 µM and 500 µM.Therefore, it is suggested from our data that either 17AAG or TMZ, at least at these drugconcentrations, are promising antitumor and radio-sensitizing agents for the managementof GBM. (a) (b) Figure 3. Radiosensitization effect of 17AAG and TMZ. (a) Surviving fraction of treating with 17AAG(24 h) and X-ray irradiation (0.57 Gy/min, 130 KV) for T98G cells; SERs for an estimated 50% survivingfraction for T98G cell line and each for 100 nM and 200 nM concentrations of 17AAG; (b) survivingfraction of treating with TMZ (24 h) and X-ray irradiation (0.57 Gy/min, 130 KV) for T98G cells; SERsfor an estimated 50% survival fraction for T98G cell line and 150 µM and 500 µM concentrationsof TMZ.Nanomaterials 2023, 13, 414 7 of 133.4. Synthesis and Characteristics of Nanofiber MeshThe spatial distribution of electro-spin NFMs was evaluated through SEM. As demon-strated in the SEM images of Figure 4a, the nanofibers have uniform diameters, and theNFM samples containing 17AAG, TMZ, or the combination of two drugs showed a ran-dom reticular arrangement and similar morphology comparable to the native blank NFM.The average diameter of most NFMs was distributed between 400–700 nm, as shown inFigure 4b.– (a) (b) —Figure 4. Nanofiber morphology and diameter under SEM. (a) SEM images of PCL-NFM; (b) averagediameters of NFM. a. Blank PCL-NFM; b. 17AAG-NFM; c. TMZ-NFM; d. 17AAG+TMZ—NFM.The tensile test was processed regarding the mechanical stability of the meshes. FromFigure S1 (in Supporting Information), the UTS of PCL-NFM was around 4.5 MPa, in thesamples of thickness 0.5 mm and width 50 mm. It was demonstrated that the mechanicalstability of the meshes is strong enough to implant into a tumor site.Moreover, we further investigated the effect of drugs on the crystallinity changeof nanofiber via DSC. As shown in Figure S2 (in Supporting Information), the meltingtemperature of PCL-NFM without TMZ (59.0 ◦C) was almost the same as that with TMZ10 wt.% (59.0 ◦C). In addition, there was no significant difference in the crystallinity changeat 37 ◦C with and without TMZ. It revealed that the mechanical properties of the meshesdo not change by adding drugs.3.5. Locally Sustained Drug ReleaseThis study aimed to demonstrate the continuous release of 17AAG and TMZ from theNFM. Figure 5 illustrates the cumulative release of 17AAG and TMZ from the NFM in PBSat pH 7.4, with a continuous release for more than 35 days. Within 24 h, the cumulativerelease rates of 17AAG and TMZ reached 28.2% and 35.8%, respectively. Therefore, it isexpected that these drugs will be distributed in relatively high concentrations in the vicinityof the NFM shortly after their implantation into the surgical cavity of the brain. During thefirst 10 days, there was also a relatively explosive release of 17AAG and TMZ. However,over the following 20 days, there was a gradual and steady trend of the cumulative releaseof both drugs. On day 35, approximately 39.7% of the TMZ and 34.1% of 17AAG werereleased from TMZ-NFM and 17AAG-NFM, respectively. The cumulative release of TMZwas slightly higher than that of 17AAG, which might be due to the slightly poorer watersolubility of 17AAG than that of TMZ. The above results suggest that our NFM releasedrelatively small concentrations of 17AAG and TMZ locally and enabled a slow, low, andsustained drug release over 35 days after an initial rapid drug release within the first 24 h.A constant drug concentration maintained locally for one month is considered one of theadvantages of local drug delivery methods, and this long-term drug release is attributed tothe unique features of the nanofiber material.Nanomaterials 2023, 13, 414 8 of 13Figure 5. Cumulative release of 17AAG and TMZ from NFM in PBS solution for 35 days.3.6. Strong Cytotoxic Effects with Nanofiber Mesh TreatmentsThe combination of radiotherapy and chemotherapy can improve the therapeutic effect,especially the local irradiation of radiotherapy can accomplish both local treatment andalleviate the systemic side effects. In addition, the molecular targeted therapy drug Hsp90inhibitor 17AAG has been shown to reduce the resistance of cancer cells to radiotherapyand chemotherapy. The synergistic effect between these different treatment modalitiesis expected to improve tumor control and allow more benefits in terms of drug dosagereduction. In the current experiment, using the NFM platform, an effort was made toinvestigate the effectiveness of radiotherapy in combination with the chemotherapeuticagent TMZ and the molecular targeted therapeutic agent 17AAG on human GBM T98Gcells. According to the results in Figure 6, the cytotoxic effects of the single-drug NFM weresignificantly enhanced when combined with 1.5 Gy X-ray irradiation. The 17AAG+TMZ-NFM showed a two-drug synergistic effect in relation to the NFM of a single agent. Thiscell-killing effect was further intensified when applied with 1.5 Gy X-ray irradiation,leading to approximately 85% of cell death, confirming the strong cell-killing effect underthe application of NFM with the simultaneous use of multiple treatments.Figure 6. The survival rates of T98G cells treated with different NFMs for 24 h with or without X-rayirradiation. (Data are mean ± SD, **** p < 0.0001, *** p <0.001, ns: not significant).Nanomaterials 2023, 13, 414 9 of 134. DiscussionGBM is one of the deadly neurological malignancies that is particularly challenging tocure because of its tumor heterogeneity, microenvironmental tumor characteristics, tumorstem-cell infiltration, and BBB properties [29]. Approximately 90% of GBM patients relapsesoon after surgery within the residual cavity or margin [30]. Standard therapy consisting ofsurgery and radiotherapy with concomitant TMZ has improved survival from less than1 year to about 15 months, but more effective therapies are still needed. In particular,high-intensity control of local lesions after surgery is essential to improve the survival ofGBM [31]. In this experiment, we propose a novel model of implanting a nanofiber systemthat combines multiple therapeutic modalities and efficient antitumor treatment while alsoexerting the effect of long-term local drug release.The idea of a surgically implanted cranial drug delivery tablet is not unique. Thecarmustine cranial tablet (GLIADEL® wafer) was approved for marketing by the FDA in1996 [32]. Carmustine (BCNU), a cytotoxic drug of the nitrosourea class, has been used inpost-surgical malignant brain tumors and continues to be utilized today [33]. The therapeu-tic efficacy and benefits are significant, while the clinical use of the GLIADEL® wafer is stillcontroversial [34]. This is primarily because, first, the greatest weakness of BNCU is theextremely short half-life of 15 min, which requires a high clinical dosage, resulting in dosetoxicity and significant side effects [35]. Second, because it is not clear how safe BCNU isin combination with other drugs, the therapeutic effect of the GLIADEL® wafer with itssingle antitumor agent only is not very effective [36]. Third, local administration provideshigher drug concentrations and fewer systemic side effects. However, previous studies onGLIADEL® wafer implantation have demonstrated the risk of local complications, such ascerebral edema and hemorrhage [37]. Although there is no evidence that polyanhydride,the material from which GLIADEL® is made, is toxic to the human body, it is believed thatsafer biomaterials than GLIADEL® wafers need to be developed and utilized to reducecomplications.In our study, NFM is made of poly-caprolactone biomaterials and contains bothTMZ, a chemotherapeutic agent with a better survival benefit than BCNU, and 17AAG, amolecular targeted agent. Temozolomide has been known to be more than 98% bioavailableand can cross the BBB [38]. Locally placed NFM containing TMZ further increases theconcentration of the drug reaching the brain tissue, preventing the brain’s protectivemechanism from expelling part of the drug out of the brain. It also has the potential toreduce gastrointestinal irritation and vomiting due to oral administration. In addition,the half-life of temozolomide, approximately 1.5 h [39], is longer than that of BCNU,which allows the concentration of the drug needed to synthesize NFM to be much lower,decreasing the probability of side effects. According to the results of this study, for T98Gcells, TMZ had a significant synergistic anticancer effect with the molecular targeted drug17AAG, which further diminished the required concentrations of both drugs and minimizedthe side effects produced by high doses of the drug as much as possible. These pointsindicate that our 17AAG+TMZ-NFM addressed some of the defects of the GLIADEL®wafer and is superior to be applied in clinical practice in the future.This experiment also considered the potential to administer radiation therapy. Postop-erative TMZ combined with radiotherapy is one of the most critical tools for the clinicalcontrol of GBM [40]. D’Alessandris et al. showed that human neural stem cells weremore sensitive to TMZ and radiation than glioma stem cells. A 30% tumor sphere madefrom glioma stem cells isolated from human GBM had an IC50 concentration of TMZ of1000 µM, more than twice higher than the IC50 measured with human neural stem cellsHNPC and NS5. Moreover, the tumor spheroids were highly resistant to a single radiationdose [41]. Therefore, in our study we focused on investigating the offsets of 17AAG tochemoresistance and radioresistance. The 17AAG is a tumor-specific, replication-dependentradiosensitizer [12]. DNA double-strand breaks (DSBs) are the leading cause of radiation-induced cell death [42]. Several studies have revealed that 17-AAG could downregulateRad51 and BRCA2 protein levels (proteins involved in the repair of DSBs), abolishing theNanomaterials 2023, 13, 414 10 of 13induction of Rad51 lesions by radiation and inhibiting the repair of double-strand breaksproduced by irradiation, leading to cell death. Moreover, this effect was specific to cancercells because 17AAG is a molecular targeted drug that binds to the Hsp90 of tumor cellswith a 100-fold higher affinity than that of normal cells [43].Multidisciplinary therapies using several modalities are becoming the standard ofcancer treatment today. However, conducting radiotherapy, chemotherapy, and moleculartargeted therapy simultaneously is challenging from the perspective of drug delivery aswell as the size and installation standards of the devices, making it difficult to achievemultiple simultaneous treatments in reality. This problem is solved by the application ofPCL-NFM. Currently, with the clinical needs, more and more nanomaterials and hydrogelmaterials are applied to local drug delivery. Compared with these materials, there are manyoutstanding advantages of PCL. It is the most-accepted synthetic polymer for medicalapplications because of its similarity to natural tissue components such as collagen fibersand ECM [44]. Additionally, as FDA-approved for use in biomedical devices, it has adiameter of 50–500 nm, excellent biocompatibility, and is biodegradable [45]. PCL is alsocost-friendly and cheap. Toxicological tests on PCL have suggested that it is non-mutagenicand non-harmful in animal models [46]. It can also be found in Figure 6 that the cellsurvival rate of blank’s PCL-NFM treatment is not significantly different from the controlgroup. PCL has a slow degradation property [47]; the most significant advantage of PCLprepared in our study is that they can release drugs in vitro for a long time (at least 35days), which allows the maintenance of local drug therapy for a significantly longer periodafter cancer surgery. In addition, PCL is easy to be processed due to its good mechanicalproperties and is usually fabricated as an extremely thin and softer sheet [48]. This makes itcompatible with the hard and soft tissues of the body, markedly mitigating the risk of poorwound healing and bleeding post-implantation. Most interestingly, a combined therapystrategy produced powerful synergistic cytotoxic effects of two drugs and a significantradiosensitization effect. These findings indicate that our NFM for GBM treatment mayprovide a new approach to developing localized medication delivery.5. ConclusionsThe 17AAG+TMZ-NFM prepared by a biodegradable poly(ε-caprolactone) materialenabled the treatment of T98G cells with simultaneous chemotherapy, radiotherapy, andmolecular targeted therapy through local sustained drug release, producing powerfulcytotoxic effects and a significant radiosensitization effect. It may become an importantapproach with promising future applications for local control of GBM.Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/nano13030414/s1, Figure S1: Mechanical property of the PCL80k NFM measured by the tensile test. Figure S2: Thermal properties of the PCL nanofibers beforeand after the addition of drugs through DSC.Author Contributions: Conceptualization, Y.M. and Y.L.; methodology, L.C.; software, Y.L.; vali-dation, Y.M., L.C., Y.S., N.F. and E.O.; formal analysis, Y.L.; investigation, Y.M., L.C., Y.S., N.F. andE.O.; resources, L.C., N.F. and E.O.; data curation, Y.L.; writing—original draft preparation, Y.L.;writing—review and editing, Y.M., L.C. and M.E; visualization, Y.M.; supervision, M.E. and H.S.;project administration, M.E. and H.S.; funding acquisition, Y.M. and M.E. All authors have read andagreed to the published version of the manuscript.Funding: This research was funded by TIA Collaboration Program Exploration and Promotion Project“KAKEHASI”, grant number TK22-026. This research was also supported by the JSPS KAKENHIGrant-in-Aid for Scientific Research (B) (JP19H04476) and the Grant-in-Aid for TransformativeResearch Areas (A) (JP20H05877).Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.https://www.mdpi.com/article/10.3390/nano13030414/s1https://www.mdpi.com/article/10.3390/nano13030414/s1Nanomaterials 2023, 13, 414 11 of 13Data Availability Statement: Data are available from the corresponding author upon request.Acknowledgments: This work was supported by JST SPRING, Grant Number JPMJSP2124, which isfor the compensation of student living expenses.Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the designof the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; orin the decision to publish the results.References1. 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Shahverdi, M.; Seifi, S.; Akbari, A.; Mohammadi, K.; Shamloo, A.; Movahhedy, M.R. Melt Electrowriting of PLA, PCL, andComposite PLA/PCL Scaffolds for Tissue Engineering Application. Sci. Rep. 2022, 12, 19935. [CrossRef]Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individualauthor(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury topeople or property resulting from any ideas, methods, instructions or products referred to in the content.http://doi.org/10.1038/s41598-022-24275-6 Introduction  Materials and Methods  Results  Drug Sensitivity Results  Synergistic Effects between TMZ and 17AAG  Radiosensitization of 17AAG and TMZ  Synthesis and Characteristics of Nanofiber Mesh  Locally Sustained Drug Release  Strong Cytotoxic Effects with Nanofiber Mesh Treatments  Discussion  Conclusions  References