Amrubicin encapsulated PLGA NPs inhibits the PI3K/AKT signaling pathway by activating PTEN and inducing apoptosis in TMZ-resistant Glioma

Glioblastoma (GBM) remains a challenging malignancy due to its aggressive nature and the lack of efficacious therapeutic interventions. Nanotechnology-based approaches exhibit promise in GBM treatment; however, the successful translation of these strategies from preclinical models to clinical settings is hindered by inefficient nanoparticle clearance from vital organs. Addressing this concern, we investigated the therapeutic potential of amrubicin (AMR) encapsulated within poly (lactic-co-glycolic acid) nanoparticles (AMR-PLGA-NPs) in combating temozolomide (TMZ) resistant GBM. The study demonstrated that AMR-PLGA-NPs exerted a pronounced inhibitory effect on the cellular viability and migratory capacity of TMZ-resistant GBM cells. Furthermore, these nanoparticles exhibited considerable efficacy in downregulating the PI3K/AKT signaling pathway, thereby inducing apoptosis specifically in TMZ-resistant glioma cells and glioma stem-like cells through the activation of PTEN. Notably, in vivo experimentation revealed the ability of AMR-PLGA-NPs to traverse biological barriers within murine models. Collectively, these findings underscore the potential therapeutic utility of AMR-PLGA-NPs as a versatile nanoplatform for addressing the formidable challenges posed by GBM, particularly in mitigating drug resistance mechanisms. The study substantiates the stability and safety profile of AMR-PLGA-NPs, positioning them as a promising avenue for combating drug resistance in GBM therapeutics.


Introduction
Gliomas represent an aggressive subset of malignancies posing substantial challenges in therapeutic management, leading to significant morbidity and mortality worldwide [1,2].Despite multimodal therapeutic interventions involving surgical resection, radiotherapy, and chemotherapy, the prognosis for glioma patients remains bleak due to the infiltrative nature of the tumor, which impedes complete surgical excision and leads to inevitable recurrence, often in the vicinity of the primary lesion or at distant intracranial sites [3].Recurrent gliomas commonly exhibit resistance to conventional chemotherapeutic agents, exacerbating the clinical dilemma and resulting in poor long-term survival rates.The efficacy of temozolomide (TMZ), an alkylating agent targeting DNA during cellular replication [4], has established it as a standard therapeutic regimen for glioma management [5].However, the emergence of TMZ-resistant gliomas poses a significant hurdle, necessitating the exploration of alternative therapeutic modalities.In this context, amrubicin (AMR), a 9-amino-anthracycline derivative with potent anticancer properties, offers promise [6].AMR exerts its effects by intercalating into DNA, inhibiting topoisomerase II activity, and disrupting DNA replication, RNA synthesis, and protein synthesis, thereby impeding cellular growth and inducing cell death.Notably, AMR exhibits enhanced antitumor efficacy compared to conventional anthracycline medications [7].To address the challenges posed by TMZ-resistant gliomas, this study focused on encapsulating AMR within poly (lactic-co-glycolic acid) (PLGA) nanoparticles (AMR-PLGA-NPs) as a potential therapeutic strategy.PLGA nanoparticles have garnered attention for their ability to traverse biological barriers, including the blood-brain barrier (BBB), owing to their small size and tunable surface properties [8].However, while PLGA nanoparticles show promise in brain drug delivery, their transport across the BBB is subject to various limitations influenced by factors such as size, charge, and composition [3].In the context of glioblastoma multiforme (GBM), the hemato-encephalic barrier (HEB) emerges as a potentially more accessible target for drug delivery compared to the BBB [9].The HEB encompasses the barrier surrounding GBM tumor cells, regulating molecular transport between the bloodstream and the brain parenchyma [10].Strategies aimed at enhancing HEB permeability, such as functionalized nanocarriers or focused ultrasound, offer potential avenues for improving drug delivery to GBM lesions, circumventing the limitations associated with BBB traversal.
The study's primary objective involved synthesizing AMR-PLGA-NPs to achieve higher drug concentrations within the brain, leveraging the favorable characteristics of PLGA nanoparticles for efficient BBB traversal.Comprehensive in vitro and in vivo assessments of AMR-PLGA-NPs in a glioma mouse model revealed negligible toxicity to vital organs.Importantly, AMR-PLGA-NPs demonstrated pronounced inhibitory effects on glioma cell migration and proliferation, enhanced apoptosis by suppressing the PI3K/AKT pathway, and exhibited the capability to breach biological barriers in TMZresistant gliomas.These pioneering findings underscore the therapeutic potential of AMR-PLGA-NPs in attenuating glioma progression, specifically in the context of TMZ-resistant tumors.The study represents a critical advancement in the pursuit of innovative glioma treatment strategies, highlighting the feasibility of leveraging nanotechnology for targeted drug delivery and overcoming drug resistance mechanisms in glioma management.

Cell culture
Dr Fawad ur Rehman of Henan University in Kaifeng, China, graciously provided the TMZ-resistant glioma cell lines U251-TMZ-R and U87-TMZ-R (U87 and U251 cells were obtained from American Type Culture Collection (Catalog # HTB-14).Cell lines were cultured in RMPI 1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (Thermo, Waltham, MA, USA) at 37 • C in a CO2 incubator.

Preparation of AMR encapsulated PLGA
PLGA nanoparticles encapsulated with AMR were prepared using a modified double-emulsion waterin-oil-in-water method as previously outlined [11,12].Initially, 100 mg of PLGA (procured from Daigang Co, Jinan, China) was dissolved in 5 mL of dichloromethane, followed by the addition of 200 µl of an aqueous solution containing 30 µg ml −1 of AMR (purchased from Simcere, China) as an internal water phase.The resulting mixture underwent sonication via a probe sonicator (Microson XL 2000, Misonix Inc., Farmingdale, NY) for 30 s for three times at 20 kHz and 30% amplitude to form the primary emulsion.The primary emulsion was then quickly mixed in 5 ml of 4% polyvinyl alcohol (PVA solution obtained from Sigma Aldrich) as an external water phase and homogenized for 30 min at 6000 rpm.After that, 5 mL of deionized water was added to the solution, and the mixture was stirred overnight at room temperature to evaporate the dichloromethane.The resulting PLGA-NPs were washed three times with double-distilled water to remove PVA residue.Subsequently, activation was achieved using 1.2 mmol of 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 1.1 mmol of N-hydroxy-succinimide (NHS, sourced from Sigma) [11].Excess EDC and NHS were removed by repetitive washing with icecold ethyl ether (Sigma).Following this, polyethyleneimine (PEI; Sigma) (PLGA-PEI; 10:1), was added drop by drop to the solution under magnetic stirring and incubated for 2 h at 20 • C. Finally, the PEI-conjugated PLGA-NPs were washed thrice with deionized water to remove excessive PEI and stored at 4 • C for further use [13].This method of preparation ensured the encapsulation of AMR within the PLGA-NPs, making them suitable for use as a drug delivery system.
The Loading of 10 ug of AMR in 1 mg PLGA-NPs was achieved via the sonication method and then the amount of AMR encapsulated in PLGA-NPs was determined by UV-Vis spectrophotometer.The determination of Total Loaded Drug Efficiency involved comparing the measured drug content in PLGA with the initially added total drug amount.During different comparisons, the concentration of AMR was kept constant, whereas the concentration of PLGA was variable.

In vitro drug release
The release of AMR from nanoparticles was conducted under two distinct conditions: phosphate buffered saline (PBS) solution with pH = 7.4 and pH = 5.0.Approximately 1 mg of AMR-loaded nanoparticles were immersed in PBS solution and agitated at 200 rpm.10 mL of supernatant was collected and measured by UV − vis spectroscopy (U-4100 Hitachi) to determine the amount of released AMR.The cumulative release of AMR from PLGA was calculated as the quantity of AMR released into the solution relative to the initially loaded amount within the PLGA nanoparticles.

Cell viability assay
The Cell Counting Kit-8 (CCK-8) assay was used to determine cell viability as per reported method [14].Briefly, TMZ-resistant glioma cells (U251-TMZ-R and U87-TMZ-R) were seeded in 96-well plates at a density of 1 × 10 3 cells/well overnight, and then treated with AMR-PLGA-NPs (6 µM) and TMZ (1 mM).After the cells had been treated for 24, 48, 72, or 96 h, 10 µl of CCK-8 solution was added to each well, and the cells were cultivated for another 2 h at 37 • C. Finally, a plate reader (Sunrise Remote; Tecan Group Ltd, Männedorf, Switzerland) was used to determine the optical density at 450 nm (OD 450).The concentration inhibiting cell growth by 50% compared with control (IC50) values for U251-TMZ-R and U87-TMZ-R cells after 1 h exposure to AMR were 6 µM ml −1 .10 µM ml −1 of TMZ was used for cell viability assay.

Colony formation assay
U251-TMZ-R and U87-TMZ-R cells (1 × 10 3 cells) were seeded in 6-well plates, allowed to grow overnight, and then AMR-PLGA-NPs and TMZ was added (1 mM).After 48 h treatment, adherent cells with normal medium were harvested for 12 d.Following this, the cells were fixed with methanol for 15 min at room temperature.After fixing, the cells are stained with 0.5% crystal violet solution at room temperature for 30 min.The plates were washed twice with tap water and dried at room temperature.A microscope (BX51, Olympus) was used to observe and count the colonies.

Migration assay
Transwell (Corning, 8 µm pore size, 24 wells) was used to assess cell migration as described previously [15].Briefly, in the lower chamber, 600 µl Dulbecco's Modified Eagle Medium (DMEM) cell culture medium (with 20% FBS) was added, while in the upper chamber, 2 × 10 5 cells (U251-TMZ-R and U87-TMZ-R) suspended in 200 µl serum-free cell culture media were loaded and in an incubator at 37 • C and 5% CO 2 for overnight.Cells on the chamber's lower surface were stained for 20 min with 0.1% crystal violet (Sigma) at room temperature.Five fields were randomly selected and imaged using a microscope (BX51, Olympus) for cell counting.

Determination of apoptosis using annexin V/PI
Annexin V/PI staining and flow cytometry analysis was performed in order to analyze the cell apoptosis.U251-TMZ-R and U87-TMZ-R cells seeded in 6-well plates were treated with AMR-PLGA-NPs and desired concentration of TMZ for 24 h.Cells were detached with trypsin (EDTA free) and collected by centrifugation.The cells were then resuspended in binding buffer for 30 min at room temperature.Next, PI (3 µl) and annexin V (5 µl) were then added to each well for 15 min at room temperature.A flow cytometer was used to detect cells, and 30 000 events were recorded.Flowjo software was used to analyze the data.

Western blotting
Western blotting was performed according to the previously reported method [3,5].In Brief, U251-TMZ-R and U87-TMZ-R cells were treated with AMR-PLGA-NPs and TMZ (1 mM) for 48 h.Total protein was extracted from cell samples using RIPA lysis buffer (Proteintech, Wuhan, China) supplemented with 50 µg ml −1 protease inhibitor PMSF after they were washed twice with PBS.The mixture was centrifuged for 30 min at 14 000 rpm at 4 • C. The protein concentration was determined using the BCA (Micro BCA protein assay kit, Thermo scientific, USA) after the supernatant was removed.Equal amounts of total protein (10 µg) were placed on 10% SDS-PAGE gels after being heated at 100 • C for 5 min.Electrophoresis was used to separate the lysates, which were then transferred onto PVDF membranes (Millipore, USA).Membranes were blocked for 1 h at room temperature in 4% BSA (Sigma-Aldrich), and then incubated with primary antibodiesovernight at 4 • C. Antibodies were purchased from the indicated companies and used at dilution 1:1000, including primary antibody: PTEN (138G6), Rabbit mAb #9559, Cell Signaling Technology (CST, USA)), Phospho-Akt (Ser473) (D9E), Rabbit mAb #4060, CST, USA), PI3 Kinase (C73F8) Rabbit mAb #4249, CST,USA), Total AKT Antibody #9272, (CST, USA), GAPDH (D16H11), Rabbit mAb #5174, CST,USA).Protein bands were seen by the electrochemiluminescence (ECL) assay kit after 1 h of incubation with horseradish peroxidase conjugated secondary antibodies (Goat Anti-Rabbit Antibody Conjugated to Horseradish Peroxidase) #1662408EDUAbbkine, China) (Bio-Rad Laboratories) at room temperature diluted as 1:1000 ratio.By measuring the band densities, the protein bands were quantified using Image J software.

Animal studies
To establish the xenograft tumor mouse model, (5 × 106 cells) U87 glioma cells suspended in 0.2 ml serum-free medium were subcutaneously injected.The cells were implanted into the lateral thoracic region of BALB/c athymic nude mice (male, 18-20 g weight, 3-4 weeks old) purchased from KeyGENBioTECH Corp., Ltd.The xenograft tumor mice were established after 4 weeks.The mice were then divided into two groups (n = 3 per group).One group received AMR-PLGA-NPs (25 mg kg −1 ) via tail vein injection, while the control group received a placebo injection of PBS.Mice were euthanized by cervical dislocation and essential organs were collected after 21 d for future study.All animal experiments were performed by following the guidelines of the National Institute of Health and Southeast University's Animal Care Research Advisory Committee (T3-HB-LAL-2023-25).The housing conditions for tumor mice models should follow the guidelines set by the Institutional Animal Care and Use Committee and the Animal Welfare Act.In general, mice were housed in clean, temperature-controlled, and well-ventilated facilities with a 12-hour light/dark cycle and appropriate bedding and enrichment.The animals were checked for pain or discomfort on a regular basis.

In Vivo BBB FL bioimaging
For the in vivo fluorescence imaging of NPs in brain, ICG encapsulated PLGA were prepared by previously reported method [8].Then, Mice were anesthetized by 3%-5% isoflurane mix with oxygen.Once the mice are properly induced and anesthetized, the isoflurane concentration was maintained to 1%-3%.Mice were injected (via the tail vein) with ICG encapsulated-PLGA, whereas control group was injected with PBS.In vivo bioimaging was performed after 24 h of injection by using IVIS Lumina FL imaging system.The FL was analyzed by the PerkinElmer software.

Statistical analysis
Statistical analyses were conducted using GraphPad Prism 7.0 to assess differences between groups, employing ANOVA followed by Tukey's test for multiple comparisons.The frequencies of cell apoptosis were compared across groups using ANOVA and Fisher LSD test.The means and standard deviation of three different experiments are provided in bar graphs.At p < 0.05, differences were considered significant in all experiments.Notably, all in vitro experiments were conducted in triplicate to ensure reliability.

Characterization of AMR-PLGA-NPs
TEM images exhibited a spherical morphology characterized by a smooth exterior surface, displaying an average diameter ranging between 100 and 125 nm (figures 1(A) and (B)).Empty PLGA-NPs and AMR-PLGA-NPs had average sizes of 83.6 nm and 125.1 nm, respectively (figures 1(C) and (D)).Additionally, the Empty PLGA-NPs presented a negative of −5.8 mV, while the AMR-PLGA-NPs displayed a zeta potential of −11.73 mV (figures 1(E) and (F)).To investigate the pH-dependent drug release of AMR, the in vitro drug release experiment was carried out at two different pH values, that is, pH = 7.4 and 5.0 as representative of physiological and cancer environment conditions [16].There is no initial burst release of AMR observed as shown in figure S1.The cumulative release of AMR from nanoparticles is less than 50% even when the experiment was carried out until 40 h.This finding suggested that the PLGA acts as a barrier to prevent premature release of drugs.

AMR-PLGA-NPs inhibit the growth of TMZ-resistant Glioma cells
Two TMZ-resistant glioma cell lines (U251-TMZ-R and U87-TMZ-R) were used and treated with AMR-PLGA-NPs and TMZ for 24-96 h to investigate the cytotoxic effect on the of glioma cells.The CCK-8 test was used to assess cell viability at the relevant concentrations and time points.As expected, AMR-PLGA-NPs treatment reduced the cell viability of TMZ-resistant Glioma cells in a timedependent manner when compared to TMZ alone and control groups (figures 2(A) and (B)).Two TMZresistant glioma stem cell lines (U251SCs-TMZ-R and U87SCs-TMZ-R) were also treated with AMR-PLGA-NPs and TMZ for 24-96 h to assess the anticancer effect of AMR-PLGA-NPs on glioma likestem cell growth.When compared to TMZ alone and the control group, AMR-PLGA-NPs treatment reduced the viability of TMZ-glioma like-stem resistant cells in a time-dependent way (figures 2(C) and (D)).Additionally, IC50 of AMR was analyzed by using CCK-8 assay at different concentration (0-10 µM) against U87-TMZ-R and U251-TMZ-R in vitro (figures S2 (A) and B).PLGA-NPs control group was not added to this study because PLGA-NPs are already reported biodegradable [17], improve therapeutic efficacy [18], with no toxicity as well as approved by the US Food and Drug Administration for medical applications [19].In particular, PLGA has been extensively studied for the development of controlled and sustained delivery of small molecule drugs, proteins and other macromolecules in commercial use and in research with respect to design and performance [3,20].

AMR-PLGA-NPs prevents colony formation of TMZ-resistant glioma cells
A colony formation experiment was performed in order to further confirm the anti-cancer activity of AMR-PLGA-NPs.In comparison to TMZ alone and the control group, AMR-PLGA-NPs decreased the clonogenicity of U87-TMZ-R and U251-TMZ-R cells (figure 3(A)-(C)).All these findings suggest that AMR-PLGA-NPs could inhibit TMZ-resistant glioma cells proliferation.

AMR-PLGA-NPs inhibits cell migration
Furthermore, we investigated the capabilities of cell migration, which is an important part of cancer growth as per reported method [21].In a transwell assay, U251-TMZ-R and U87-TMZ-R cell migration was considerably reduced in the AMR-PLGA-NPs treated group compared to the TMZ treated and control groups, as shown in figures 4(A)-(C).

AMR-PLGA-NPs induced cell death by PI3K/AKT signaling pathway
The effect of AMR-PLGA-NPs on U251-TMZ-R and U87-TMZ-R cells was investigated to validate whether apoptosis is involved or not.Flow cytometry was used to evaluate the proportion of apoptotic cells by labelling cells with PI and annexin V-FITC.In comparison to the control, AMR-PLGA-NPs caused a significant increase in apoptosis (figures 5(A)-(D)).
Furthermore, in our study, we aimed to investigate the impact of AMR-PLGA-NPs on the PI3K/AKT pathway, which plays a crucial role in cancer development and progression.We focused on evaluating the effects of AMR-PLGA-NPs in TMZ-resistant glioma cells.To assess the changes in pathway activity, we performed western blot analysis using anti-PI3K and anti-phosphorylated AKT antibodies.Our results revealed notable alterations in the PI3K/AKT pathway upon treatment with AMR-PLGA-NPs.Specifically; we observed a significant reduction in the protein level of PI3K as well as a decrease in AKT phosphorylation in U251-TMZ-R and U87-TMZ-R cells.These findings highlight the potential of AMR-PLGA-NPs as a promising therapeutic approach for cancer treatment by targeting and modulating the PI3K/AKT signaling pathway.Notably, the expression of total AKT protein remained unaffected.Because PTEN is a well-known upstream suppressor of PI3K/AKT signaling [22], we next examined whether PTEN was involved in the control of PI3K/AKT signaling by AMR-PLGA-NPs.The treatment of U251-TMZ-R and U87-TMZ-R cells with AMR-PLGA-NPs resulted in overexpression of PTEN at the protein level (figures 6(A)-(D)).All of these data point to the possibility that AMR-PLGA-NPs influence apoptosis by blocking the PI3K/AKT signaling pathway via activation of PTEN.

Discussion
Gliomas are brain tumors that frequently recur and become resistant to the chemotherapy drug TMZ, which poses a significant challenge in their treatment   [ 3,23].Treatment options for recurrent gliomas are notably limited, offering constrained outcomes [21].Therefore, the researchers directed their focus toward TMZ-resistant gliomas, aiming to discover a therapy capable of curbing tumor progression or eliminating TMZ-resistant glioma cells, either as a standalone treatment or in combination with other drugs.The study's findings suggest that AMR-PLGA-NPs have the potential to achieve this goal.
The study employed CCK-8 and colony formation assays to evaluate the impact of AMR-PLGA-NPs on the viability and clonogenicity of TMZ-resistant glioma cells.The results indicated that AMR-PLGA-NPs exert a potent anticancer effect on these cells, significantly reducing their viability and clonogenic potential.Additionally, the investigation of apoptosis, a critical indicator in anticancer therapy, revealed a substantial increase in the percentage of apoptotic cells following treatment with AMR-PLGA-NPs.
The PI3K/AKT pathway has been shown to play a significant role in the development of various malignancies, including glioma [24,25].AKT is a downstream target of PI3K, and its active type phosphorylation regulates several cellular processes such as proliferation, cell growth [26], the cell cycle, apoptosis, and protein synthesis [27].PTEN is a negative regulator of the PI3K/AKT pathway and inhibits cell growth and proliferation [28].To investig-ate the effect of AMR-PLGA-NPs on the PI3K/AKT signal pathway, the study assessed the expression of PTEN, PI3K, phosphorylated AKT.The findings revealed that treating TMZ-resistant glioma cells with AMR-PLGA-NPs increased PTEN expression while decreasing PI3K and phosphorylated AKT expression.Consequently, the overexpression of PTEN, which suppresses the PI3K/AKT pathway, could be a unique molecular effect of AMR-PLGA-NPs in TMZresistant glioma.
The in vivo efficacy of AMR-PLGA-NPs was evaluated using BALB/c athymic nude mice, and the results demonstrated significant inhibition of tumor growth compared to the control group treated with PBS.Moreover, the tumor growth curve in the AMR-PLGA-NPs group displayed a notable suppression trend in comparison to the control group (figure 7(C)).In vivo fluorescence imaging results illustrated the targeted and rapid accumulation of ICG-AMR-PLGA-NPs in the brain region, supporting the ability of PLGA to penetrate the BBB.
These findings suggest that AMR-PLGA-NPs exhibit pronounced inhibitory effects on tumor growth in vivo, without causing significant changes in body weight.Additionally, the efficient accumulation of ICG-AMR-PLGA-NPs in the brain region indicates the potential of PLGA as a carrier for targeted drug delivery across the BBB.

Conclusion
In summary, our findings demonstrated that AMR-PLGA-NPs induce apoptosis in human TMZresistant glioma cells, effectively restraining their proliferation.Furthermore, our study revealed that AMR-PLGA-NPs upregulated PTEN expression while concurrently inhibiting the PI3K/AKT signaling pathway.This suggests a sugnificant mechanism through which AMR-PLGA-NPs impede the proliferation of TMZ-resistant glioma cells.Moreover, our investigation exhibited that AMR-PLGA-NPs not only inhibited tumor growth in an in vivo TMZresistant glioma xenograft model but also exhibited targeted delivery and accumulated in the brain region.This targeted drug delivery approach minimizes systemic side effects, maximizes drug concentration at the tumor site.Furthermore, the utilization of PLGA-based drug delivery systems facilitates sustained release of therapeutic agents over an extended period sustained release of therapeutic agents over an extended period.This helps maintain therapeutic drug levels in the tumor region, reducing the need for frequent dosing and improving treatment efficacy.Furthermore, the direct delivery of drugs to the tumor site by AMR-PLGA-NPs offers the potential to reduce the likelihood of drug resistance developmenta notable concern in glioma therapy.This targeted delivery strategy presents a promising opportunity for addressing challenges associated with drug resistance development in glioma treatment.

3. 6 .
AMR-PLGA-NPs inhibit tumor development in vivo To see if AMR-PLGA-NPs have similar effects in vivo, BALB/c athymic nude mice were employed.When compared to xenografts treated with PBS, xenografts treated with AMR-PLGA-NPs developed at a significantly slower rate (figures 7(A) and (B)).In comparison to the control group, the tumor growth curve in AMR-PLGA-NPs treated mice displayed a relatively modest tendency (figure 7(C)).The weight of mice in the treatment and control groups did not differ significantly on any of the days tested (figure 7(D)).In order to determine the ability of PLGA to penetrate the BBB, we conducted in vivo fluorescence imaging.The results presented demonstrate the targeted and swift accumulation of ICG-AMR-PLGA-NPs in the brain region, as depicted in figure 7(E).

Figure 4 .
Figure 4. Glioma cell migration was measured using a transwell assay.In comparison to TMZ alone and control, U251-TMZ-R and U87-TMZ-R cells displayed lower migratory ability after culture with AMR-PLGA-NPs (A).Scale bar: 100 mm.The graph depicts the mean and standard deviation of migrating cells from three separate trials (B), (C).( * * * p < 0.001 and * * * * p < 0.0001).

Figure 7 .
Figure 7. xenograft model in vivo.Effect of AMR-PLGA-NPs was also evaluated in xenograft tumor mice model (A).Graphs showing the final tumor weight, volume, and body weight (B-D) after administration of NPs ( * * P <0.01).In vivo fluorescence imaging to assess the targeted and rapid accumulation of ICG-AMR-PLGA-NPs in brain region (E).