A type of brain-targeting nano-formulation and its anti-tumour effect in paediatric brain tumour cells

ABSTRACT

In this study, we grafted cholesterol with pullulan to synthesize hydrophobic pullulan(CHP). Three CHP polymers (CHP1, CHP2, and CHP3), with different degrees of substitution were designed by changing the feed ratio of cholesterol to pullulan. The ¹H-NMR results showed that the hydrophobic substitutability of CHP1, CHP2, and CHP3 were calculated to be 8.26%, 5.67%, and 3.52%, respectively. The sizes of CHP1 NPs, CHP2 NPs, and CHP3 NPs were (118±7.9), (123±4.7) and (158±9.3) nm, respectively. The CHP NPs embedded with VCR and adsorbed Tween 80 (CHPT@VCR NPs). The results of microscale thermal imaging (MST) testing indicated the CHP-1 NPs with the highest degree of cholesterol substitution possessed the highest adsorption capacity for Tween 80. In vitro pharmacological studies were conducted in SJ-GBM2 cells, the results showed that the tumor cells inhibition rate and the cellular uptake rate of CHPT@VCR-1 NPs were the highest compared with CHPT@VCR-2 NPs and CHPT@VCR-3 NPs.

KEYWORDS:

• Pediatric brain tumours
• cholesterol
• pullulan
• Tween 80
• vincristine

Introduction

Paediatric brain tumour is a common neurological disease in children, with the highest incidence among solid tumours. Brain tumours are the leading causes of death in paediatric tumour patients and a serious threat to children’s health [1,2]. The treatments of paediatric brain tumours involves surgical resection, radiation therapy, adjuvant chemotherapy and immunotherapy in clinical practice [3–5]. The infiltrative nature of malignant brain tumours makes it impossible for patients with brain tumours to be cured by radical surgery. Chemotherapy has become a treatment option for controlling residual and micro-metastatic tumours that cannot be surgically removed. The blood–brain barrier (BBB) can protect the brain from harmful macromolecules and pathogens in the blood circulation, but it can hinder drug penetration, leading to suboptimal drug concentrations in the central nervous system (CNS) [6,7]. Hydrophilic stabilizers such as polysorbate have been found to modify nanoparticles and endow them with long-term cycling properties [8,9]. Hydrophilic stabilizers produce chain clouds after coating the surface of hydrophobic polymers, which will repel plasma proteins and avoid being recognized by regulatory factors and engulfed by macrophages in the mononuclear phagocytic system (MPS) [10–12]. In recent years, Tween 80 has been widely studied in the treatment of brain diseases due to its amphiphilicity and low toxicity. It was explored to modify the surface of nanocarriers for the delivery of a number of the CNS drugs into the brain of animals with varying success. Nanoparticles coated with Tween 80 can preferentially adsorb apolipoprotein E (ApoE) on their surfaces, using the high affinity of ApoE for the low-density lipoprotein receptor (LDLr) and endothelial cells [13,14], crossing the BBB through endocytosis to achieve active brain targeting of nanodrug preparations and improve drug concentration and bioavailability in brain tissues [15]. Nanoparticles coupled with Tween 80 has shown significant therapeutic effects in the treatment of Alzheimer’s disease, and the result of in vitro fluorescence imaging showed that the nanoparticles have successfully targeted the brain, reduced accumulation in the liver [16]. In addition, Tween 80 coated nanoparticles are feasible in the treatment of intracranial infections and brain tumours [17–19].

Vincristine (VCR) is a commonly used natural plant-based anticancer drug in clinical practice, which anti-tumour mechanism is to inhibit the aggregation of microtubule proteins, prevent the formation of spindle microtubules and stop cancer cell mitosis midway through the process [20]. However, due to its low stability, easy decomposition, low solubility and difficulty in penetrating the BBB, it is difficult to achieve effective concentration in brain tissue, which limits its application in the treatment of malignant paediatric brain tumour [21]. The structural and functional differences between tumour blood vessels, tumour lymphatic system and normal tissues and poor connections between endothelial cells provide a pathway for the infiltration of nanoparticles. The loss of lymphatic system function reduces the recovery of nanoparticles, allowing them to remain in tumour tissue [22,23]. Therefore, VCR can be loaded in nanomaterials to form a nanomedicine delivery system, which can not only improve the solubility of drugs but also achieve passive targeting by enhancing the permeability and retention effect (EPR effect) of tumour tissue and achieve large accumulation and slow controllable release at the tumour site to reduce toxicity and side effects.

Various drug carriers have been developed and reported in the literature until now [24]. Bioactive agent carriers synthesized from polymer materials (such as hydrogels, microparticles and nanoparticles) have been widely used in many nanomaterial fields due to their strong biodegradability and low toxicity of natural polymers, such as anti-bacterial [25,26], anti-cancer [27,28], tissue engineering with biosensors and bioinformatics [29,30]. Pullulan is a naturally degradable polymer with advantages such as safety, non toxicity, good biocompatibility and easy modification. Pullulan contains multiple hydroxyl groups in each gum unit, which can be used to connect the strong hydrophobic group cholesterol to the main chain through esterification reactions, generating amphiphilic and hydrophobic modified pullulan polysaccharide polymers (CHP) [31,32]. The polymer can self assemble into nanoparticles with a unique ‘core-shell structure’ through the cohesiveness of the hydrophobic part in aqueous solution, and load small molecule drugs onto their hydrophobic centres to form pullulan nanomedicine formulations [33,34]. This nanodrug formulation not only has a strong solubilizing effect on insoluble drugs but can also be slowly released in vivo, undergo long-term circulation and controlled release in the acidic microenvironment of the tumour issue [35].

The efficacy of nanomedicine formulations is influenced by multiple factors. In previous studies, we explored the relationship between surface modification of nanoparticles and their functions [32,36]. Based on these results, it is known that in addition to surface modification affecting the receptor ligand affinity of nanoparticles, hydrophobic substitution also affects the performance of nanoparticles by affecting their particle size, drug release and cell targeting ability. Naturally, the larger the degree of hydrophobic substitution, the smaller the size of the nanoparticles, which are more easily absorbed by cells and reduce the release of free drugs during plasma transport, resulting in better sustained release. Meanwhile, due to the different capillary pores of different types of tumours, the generation of EPR effect requires the particle size of the nanoparticle to be compatible with the pore size of the capillary wall. Oversized particles cannot penetrate through capillaries and penetrate into tumour tissue [37], whereas undersized particles are easily removed by various internal tissues and cannot be effectively retained in tumour tissue [22]. Therefore, in order to choose the appropriate particle size for a specific type of tumour, it is necessary to balance the ability to penetrate the tumour (reducing particle size) with reducing toxicity in normal tissues and increasing clearance of the reticuloendothelial system (increasing particle size). In this study, we prepared three pullulan cholesterol polymer nanomaterials coated with Tween 80 with different degrees of hydrophobic substitution for encapsulating VCR. The nanomaterial was designed for targeted therapy of brain tumours, providing new insights into the relationship between the degree of hydrophobic substitution of nanoparticles and therapeutic efficacy, and will also be beneficial for the development of brain targeted nanodelivery systems.

Materials and methods

Experimental reagent and instrument

Polysorbate-80 (Tianjin Chenfu Reagent Research Institute) and VCR (Chinese Aladdin reagent) were used. The remaining reagents were domestic analytical reagents. A constant temperature magnetic stirrer (IKA RCT basic, Germany), vacuum freeze dryer (Maxi Dry Lyo, Heto Holten), Transmission Electron Microscopy (TEM, Japan JIEM-100S), Dynamic Light Scattering Instrument (DLS, British Malvern ZS-90), ultraviolet–visible spectrophotometer (JASCO V-560, U.S.A.) and isothermal titration calorimeter (VP-ITC, Microcal Inc., Northampton, Massachusetts) were used. SJ-GBM2 cells (children brain tumour glial cells) from the American Type Culture Collection (ATCC) cell bank (Manassas, Virginia, U.S.A.) and C57BL/6 mice (Hunan Slack Jingda Laboratory Animal Co., Ltd.) were used. The animal experiments of this study were conducted in the Hunan Provincial Key Laboratory of Small Molecule Targeted Drug Research and Creation, School of Medicine, Hunan Normal University, approved by the Institutional Animal Care and Use Committee, School of Medicine, Hunan Normal University (IACUC protocol number: 2023193).

Synthesis of CHP polymers

A total of 0.5 g of pullulan sample were dissolved in 15 mL of dimethyl sulphoxide (DMSO) without water, and a certain amount of cholesteryl succinic anhydride (CHS), 4-dimethylaminopyridine (DMAP) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were added. The solution was dissolved in 10 mL DMSO with a feeding ratio of DMAP:CHS = 1:1 and EDC: CHS = 1.2:1, stirred and reacted (room temperature) for 1 h. Then, the CHS and sugar units of the reaction solution were used, and pullulan solution-to-feed ratios of 1:5, 3:20 and 1:20 (mmol/mmol) were used as the ratios of the mixed reaction. The reaction solution was dropped into 200 mL of absolute ethanol to form a white precipitate, which was filtered with suction. The product was washed with an appropriate amount of ethanol, tetrahydrofuran and ether and dried at 80°C to obtain the CHP1, CHP2 and CHP3 polymers, respectively (Figure 1).

Figure 1. Synthesis route of CHP1, CHP2 and CHP3.

¹H NMR of CHP polymers

The CHP1, CHP2 and CHP3 polymers were dissolved in DMSO-d₆, and CHS was dissolved in CDCL₃-d₆. The sample¹H NMR spectra were obtained for analysis. By using its NMR spectrum, the degree of substitution of cholesterol in the CHP polymer was determined by the peaks representing the α-1,4 glycosidic bond and the α-1,6 glycosidic bond and the area under the methylene peak.

Preparation of CHP NPs

Twenty milligrams of CHP1, CHP2 and CHP3 polymers were added to 1 mL DMSO to dissolve them. The solution was transferred to a dialysis bag and placed into 3 L of distilled water. After dialysis was completed, the mixture was filtered with a 0.45 μm microporous membrane to prepare CHP1, CHP2 and CHP3 NPs with three degrees of substitution. The volume of CHP NPs was adjusted to 2 mg/mL.

Preparation of drug-loaded nanoparticles (CHP@VCR)

Four milligrams VCR and twenty milligrams CHP1, CHP2 and CHP3 polymers were dissolved in the mixture of DMSO and triethylamine (TEA/VCR = 2:1, mmol/mmol); the mixture ratio of the drug and material solution is 1:5. Put them into a dialysis bag (MWCO = 8000–14000 Da) and dialysis with water for 24 hours. When dialysis was finished, the remaining solution was treated with a 50 W ultrasound probe for 2 minutes, transferred in a 10 mL volumetric flask and filtered with a 0.45 μm filter to obtain CHP@VCR.

Preparation of CHPT@VCR nanoparticles

A certain amount of prepared CHP@VCR nanoparticles were placed into a 10 mL beaker and pulled into a beaker containing a certain concentration of polysorbate 80 emulsifier. After the two were allowed to stand for 1 h, the mixed solution was placed in an EP tube. Then, ultrasonic treatment was performed with a probe for 3 min, and the operation was repeated three times. The mixture was drawn with a syringe and filtered to remove impurities through a filter membrane to obtain CHPT@VCR nanoparticles. Preparation of nanoparticles loaded with fluorescent substances: Following the same method as for the encapsulated drugs, we loaded the fluorescent substances ICG and FITC on CHPT@VCR nanoparticles for subsequent in vivo fluorescence imaging and cellular uptake, respectively.

Characterization of CHP, CHP@VCR and CHPT@VCR nanoparticles

The prepared CHP, CHP@VCR and emulsified CHP@VCR nanomicelles were placed into the cuvette, and the cuvette was placed into the sample chamber of the DLS particle size analyser for testing. Each sample was tested three times. The test conditions were as follows: argon ion laser, wavelength 658 nm, temperature, 25 ± 0.1 degrees, dynamic light scattering angle, 90 degrees. For simultaneous determination of the zeta potential, the operating conditions were 11.4 v/cm, 13.0 mA, and 25°C, and the sample solvent was diluted with distilled water. The nanomicelles were dropped on a copper mesh coated with a carbon support film, stained with 2% phosphoric acid and dried naturally, and their morphology was observed under TEM.

Encapsulation efficiency, drug loading and drug release

0.1 g of VCR was dissolved in 50 mL of water, and the dilution method was used to prepare solutions of 1 μg/mL, 2 μg/mL, 3 μg/mL, 4 μg/mL and 5 μg/mL. The UV absorbance at 297 nm was measured to draw a standard curve.

The volume of CHP@VCR nanomicelles and CHPT@VCR nanomicelles was kept constant in a 10 mL volumetric flask, 0.2 mL of DMSO was added, and the solution was then sonicated for 2 minutes. The UV absorbance was measured at 297 nm and was brought into the value obtained from the standard equation, and the drug loading efficiency (LC%) and encapsulation efficiency (EE%) were calculated.

To detect drug release, we adopted the hydrodynamic dialysis method. Two millilitres of CHPT@VCR-1 NPs, CHPT@VCR-2 NPs, CHPT@VCR-3 NPs and VCR-Tween 80 were placed in a dialysis bag (3500 Da) and dialysed on a 37°C constant temperature shaker (100 rpm) containing 50 mL of PBS (pH 7.4 or pH 6.8). At 0, 0.5, 1, 2, 3, 4, 8, 12, 24 and 48 hours, the entire PBS release solution was replaced with fresh PBS release solution. PBS was collected at specific time points (t), the volume of PBS was determined(V_t), the absorption at 297 nm was measured, and the concentration of the drug was calculated as C_t.

$\mathit{Q}\mathrm{\%}=\frac{{\mathit{V}}_0{C}_0+\sum _{\mathit{t}=0}^n{V}_{\mathit{t}}{C}_{\mathit{t}}}{V_{\mathit{db}}{C}_{\mathit{db}(\mathit{drug})}}\times 100\mathrm{\%}$

Specifically, Q is the drug release rate at time t (hours)(t = 0, 0.5, 1, … n … 48; V₀ and C₀ are equal to 0); V_db is the volume of PBS in the dialysis bag; and C_db (drug) is the initial concentration of nanomaterials.

Microscopic thermophoresis (MST) analysis

All MST experiments were carried out on the integral NT.115 system. All solutions were prepared with deionized water and analytical reagents. The buffer was prepared and stored at room temperature. The protein sample was kept refrigerated until use. The three types of CHP NPs were diluted to 40 nM with deionized water and loaded with Tween 80 for fluorescence detection. Tween 80 solution was prepared. Sixteen capillary tubes were labelled 1–16; first, 20 μL of Tween 80 was added to tube 1, and 10 μL was added to tube 2 to 16. Then, 10 μL of the solution was transferred from tube 1 to tube 2 and mixed well. After that, 10 μL of the solution was removed from tube 2 and transferred to tube 3. This operation was repeated until 10 μL of solution was finally removed from tube 16 to ensure that the volume of the solution in each tube was the same. One microlitre of diluted CHP pellets was added to each test tube and mixed well to start the measurement. NT analysis software was used to analyse the MST test data, and KD fitting was performed according to the law of mass action according to the software instructions.

Cell culture and experimental design

The purchased SJ-GBM2 cell line was routinely cultured in DMEM supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) in a humidified incubator at 37°C and 5% CO₂.

Cellular uptake

SJ-GBM2 cells were seeded into 24-well plates (1 × 10⁵ cells/mL). FITC loaded CPHT@VCR nanoparticles were added to the cells when they reached 70% confluence. DIPA (1 μg/mL) was used to stain the cells, photos were taken at 2 hours, and using Image J software to analysis.

Cytotoxicity of nanoparticles

The cytotoxicity test was carried out by the MTT method. SJ-GBM2 cells were seeded into 96-well plates (5000 cells/well). Different concentrations of VCR, CHPT@VCR-1 NPs, CHPT@VCR-2 NPs and CHPT@VCR-3 NPs were cultured with the cells for 48 hours. Then, 20 μL MTT solution (5 mg/mL) was added to each well, incubation solution was aspirated 4 hours later, 150 μL DMSO was added to each well, the 96-well plate was placed in a multi-functional microplate reader at room temperature and shaken for 10 min, the absorbance of each well was measured at a wavelength of 570 nm and the cell survival rate was calculated.

Cell migration experiment

A total of 8 × 10⁵ cells were seeded in 6-well plates until fully confluent. The three formulations of CHPT@VCR were dipped with a glass rod, and scratches were made on the surface; the cells were cultured with serum-free DMEM. Digital images were taken at 0, 12, 24 hours. Image J was used to calculate the average area, and the experiment was repeated three times.

Experimental design of brain targeting

In vivo fluorescence imaging technology to observe brain-targeting effects: C57BL/6 mice (18 g–22 g) was used as the experimental object. The mice were randomly divided into three groups, and 200 μL of free ICG, ICG loaded CHPT@VCR-1 and CHP-VCR nanoparticles (concentration of ICG is 200 μg/mL) were injected through the tail vein. Live fluorescence imaging of mice was taken after 0.5 hours.

Statistical analysis

The experimental results are expressed as the mean ± standard deviation (SD). The statistical significance of differences between the means of each group was analysed by one-way ANOVA, followed by Tukey’s multiple comparison test using GraphPad Prism 6.0. Sample size (n) for each statistical analysis is 3. P-values are usually expressed as p > 0.05 to indicate insignificant differences; 0.01< p < 0.05 indicate significant differences; p < 0.01 indicates extremely significant differences.

Results

Preparation and characterization of CHP

To demonstrate that the synthesis of the nanomaterials was successful, we examined the ¹H-NMR spectra of the CHP. As shown in Figure 2, the ¹H NMR spectrum of pullulan consisted of peaks from 0.60 to 2.40 ppm (the hydrogen on the cholesterol skeleton) and hydrogen peaks on the sugar unit at 2.60 ~ 4.60 ppm (2 H, 3 H, 4 H, 5 H, 6 H), 4.60 ppm [l H α (1–6)], 5.05 ppm (l H α (1–4)) and a 4.60 ~ 5.40 ppm hydroxyl group. CHP showed an NMR peak of 2.54 ppm (-OCH₂CH₂O-) that was absent from the NMR spectrum of pullulan, which confirmed that CHS was successfully connected to pullulan (Figure 2). The 2.54 ppm (4 H, CHS-OCH₂CH₂O-) peak in the CHP spectrum was easy to distinguish, and the peak area ratio could be used to calculate the degree of substitution (DS) of cholesterol on pullulan using the following formula:

$\mathit{DS}\%=\frac{{\mathit{A}}_{\mathrm{\partial }2.53}}{4(A_{\mathrm{\partial }4.68}+A_{\mathrm{\partial }5.00)}}\times 100\mathrm{\%}$

Figure 2. ¹H NMR spectra of pullulan and three types of CHP nanoparticles.

DS of cholesterol was 8.26%, 5.67% and 3.52% for CHP1, CHP2 and CHP3, respectively.

The average particle diameters of CHP-1, CHP-2 and CHP-3 nanoparticles measured by DLS were 118 ± 7.9, 123 ± 4.7 and 158 ± 9.3 nm, respectively, and the nanopolymer dispersion index (PDI) values were 0.189 ± 0.039, 0.156 ± 0.048 and 0.119 ± 0.023, respectively (Figure 3). The zeta potentials of the CHP NPs were all approximately 0 and in the range of −1 to 1 mV. Under the electron microscope, CHP self-aggregates into a spherical nanostructure with hydrophilic groups as the shell and hydrophobic groups as the core with a regular shape.

Figure 3. The sizes, zeta potentials and TEM morphology of three CHP nanoparticles.

Characterization of CHP@VCR and CHPT@VCR nanoparticles

As shown in Figure 4, the nanoparticle diameters of CHP@VCR-1, CHP@VCR- 2 and CHP@VCR-3 NPs were 178 ± 4.8 nm, 218 ± 7.2 nm and 280 ± 2.7 nm, and the dispersion index (PDI) values were 0.196 ± 0.023, 0.189 ± 0.020 and 0.154 ± 0.038, respectively. The particle size increased with the decrease in the degree of hydrophobic substitution, but the magnitude of the size change in the CHP formulated nanoparticles before and after drug loading was larger than that of the blank particles, indicating that the nanoparticles had different loading capacities for VCR. After the drug is administered, the degree of hydrophobic substitution enhances the influence of the nanoparticle particle size. The dispersion index (PDI) values of the three drug-loaded nanoparticles were 0.196, 0.189 and 0.154, indicating that the nanoparticles had good dispersibility in aqueous solutions. The zeta potential values of the three nanoparticles were −1.8 ± 0.3 mV, −1.5 ± 0.2 mV and −1.4 ± 0.5 mV, respectively. The nanoparticle diameters of CHPT@VCR-1 NPs, CHPT@VCR-2 NPs and CHPT@VCR-3 NPs were 188 ± 3.4 nm, 238 ± 7.8 nm and 307 ± 9.8 nm, respectively. The zeta potential values of the three kinds of nanoparticles were −1.6 ± 0.1 mV, −1.0 ± 0.3 mV and −0.6 ± 0.3 mV, respectively.

Figure 4. Particle size diagram of three feeding ratios of CHP@VCR NPs (1), CHPT@VCR NPs (3) and potential diagram of three feeding ratios of CHP@VCR NPs (2) and CHPT@VCR NPs (4).

MST analysis to explore the adsorption of CHP on Tween 80

MO affinity analysis software is used to analyse the collected data. The MO control software automatically checked the initial fluorescence of all capillaries before measuring MST (Figure 5a) and started analysing the MST signal when the fluorescence changed within 20%. The KD model was chosen to obtain the dose–response curve. To display the data, the option score boundary was selected for normalization (Figure 5b). Divide all F-modulus values of the curve by the curve amplitude to obtain the score limit for each data point (from 0 to 1). The method is independent of the starting level and amplitude of the F-norm, thus allowing for the comparison of Kd values for interactions with very different amplitudes. By using MO software, consistency between repeated experiments can also be evaluated by combining all experimental replicates. The experiment was conducted three times. The Kd values of Tween 80 binding to all three CHP studied were determined, and the affinity of three CHPs with Tween 80% was analysed. Among them, CHP1 has the highest affinity for Tween 80 (255 μM), next is CHP2 (388 μM) and CHP3 (521 μM) (Figure 5c).

Figure 5. Capillary fluorescence scan (a); MST trajectories of three feeding ratios of CHP NPs (b); dose‒response curves of three feeding ratios of CHP NPs interacting with Tween 80 (c).

Figure 6. Drug release profiles of three drug-loaded CHPT NPs and VCR-adsorbed Tween 80 at pH 6.8 (a) and pH 7.4 (b).

Drug loading and drug release

We measured the drug loading of the nanoparticles and drug release from the nanoparticles. The drug loading of VCR into CHPT@VCR-1 NPs, CHPT@VCR-2 NPs and CHPT@VCR-3 NPs were calculated to be approximately 7.25 ± 0.42%, 6.58 ± 0.36% and 5.86 ± 0.29% by UV–Vis spectroscopy, and EE were 78.28 ± 2.24%, 71.76 ± 1.89% and 62.50 ± 1.56%, respectively. Next, we conducted drug release experiments to evaluate the sustained release of the drugs from the nanoparticles and their tumour microenvironment-endosome responsiveness. The result in Figure 6 showed that the drug release rates of CHPT@VCR-1 NPs, CHPT@VCR-2 NPs and CHPT@VCR-3 NPs were 21.48 ± 2.23%, 30.36 ± 2.37% and 37.58 ± 3.55% in PBS at pH 7.4 for 48 hours. All three nanoparticles exhibited sustained-release properties, the stronger the hydrophobicity was, the slower the drug release rate was. Compared with the neutral environment, in the simulated tumour endosome microenvironment, the VCR release rates from CHPT@VCR-1 NPs, CHPT@VCR-2 NPs and CHPT@VCR-3 NPs were 42.53 ± 2.30%, 52.68 ± 2.41% and 57.36 ± 2.39%, respectively, which was significantly increased. The slow-release control of the CHPT@VCR-2 NPs and the CHPT@VCR-3 NPs was not as obvious as that of CHPT@VCR-1 in both neutral and tumour slightly acidic environments, which suggested that the hydrophobic groups of CHPT@VCR NPs. The results showed that (Figure 8) with increasing of VCR concentration, the proliferation activity of SJ-GBM2 cells gradually decreased. CHPT@VCR-2 NPs and CHPT@VCR-3 NPs may could not be used as carriers to achieve the expected sustained-release effect.

Figure 7. Cell survival rate graph of SJ-GBM2 cells under three groups of CHPT@VCR NPs and free VCR treatments (**p < 0.01, ****p < 0.0001).

In vitro antitumor activity of CHPT@VCR nanoparticles

Next, we investigated the inhibitory effect of three CHPT@VCR nanoparticles on SJ-GBM2 cells. We use MTT to detect cell proliferation after drug treatment. The results in Figure 7 showed that with VCR concentration increasing, the proliferation activity of SJ-GBM2 cells gradually decreased. Compare to free VCR treatment group, CHPT@VCR-1 NPs showed higher cell viability inhibition rate on SJ-GBM2 cells. When the VCR concentration reaches 0.818 μg/mL, the tumour cell survival rate treated with CHPT@VCR-1 NPs was the lowest at 28.7 ± 6.23%. This result showed that the inhibitory effect of CHPT@VCR-1 NPs on SJ-GBM2 cell survival was more significant than that of the other two nanoparticle formulations.

To further explore the mechanism by which CHPT@VCR nanoparticles enhance the cytotoxicity of the drugs, we performed cellular uptake experiments. As shown in Figure 8, three CHPT@VCR nanoparticles were significantly ingested by cells at 2 hours, and CHPT@VCR-1 NPs exhibited the strongest cellular uptake efficiency in the three groups of nanoparticles. Nanoparticles are rapidly taken up by cells in our experiments, which significantly increases intracellular drug concentration, thereby improving drug efficiency. To investigate the role of the nanoparticles in tumour migration, scratch experiments were performed (Figure 9). After 12 hours of administration, the migration rates of SJ-GBM2 cells treated with CHPT@VCR-1 NPs, CHPT@VCR-2 NPs, CHPT@VCR-3 NPs and blank nanoparticles were 32.57 ± 1.22%, 38.23 ± 1.24%, 71.32 ± 1.9% and 80.13 ± 1.93%, respectively. Among them, CHPT@VCR-1 NPs showed the highest inhibitory migration rate. The cells in the control group maintained their original migration ability and covered scratches by migrating, while the cell migration ability in the CHPT@VCR group was inhibited, which proved that nanoparticles we prepared can be continuously absorbed by cells in vitro and inhibit tumour growth while reducing tumour migration, and CHPT@VCR-1 NPs have significant potential in inhibiting tumour growth.

Figure 8. Uptake of FITC-labeled CHPT@VCR NPs at 2 hours in SJ-GBM2 cells, green for FITC(2 μg/mL), blue for DAPI(1 μg/mL).

Figure 9. Cell migration diagram of three CHPT@VCR NPs (a); cell migration rate of three CHPT@VCR NPs groups (b) (**p < 0.01, ***p < 0.001 and ****p < 0.0001).

In vivo fluorescence imaging assessment of brain targeting

Near-infrared-II (NIR-II) fluorescence is typically preferred for in vivo fluorescence imaging due to its low spontaneous fluorescence from surrounding tissues and good tissue penetration [38]. As an optical diagnostic agent, indocyanine green (ICG) is a medical diagnostic dye approved by the US Food and Drug Administration (FDA) for measuring cardiac output, liver function and ophthalmic angiography [39]. Compared with endogenous pigment clusters such as blood and melanin, ICG has higher absorption capacity, making it widely used in photoacoustic imaging. For example, the anticancer drug doxorubicin (DOX) and the NIR dye ICG have been successfully encapsulated into thermosensitive liposomes based on natural phase change materials, which can indicate the distribution of liposome nanoparticles in animals [40]. To investigate the brain targeting of nanoparticles, we used ICG as a fluorescent agent to indicate the in vivo distribution of nanoparticles, and captured fluorescence images of the entire body of living mice 0.5 hours after injection (Figure 10). The results showed that free ICG did not show fluorescence in the mouse brain; CHPT@VCR-1 NPs and CHP@VCR-1 nanoparticles showed obvious brain targeting, and CHPT@VCR-1 nanoparticles are much stronger. In conclusion, the result indicated that the drug loaded nanoparticles can reach the brain through the tail vein and have corresponding targeting properties, and the adsorption of Tween 80 on the surface of the nanoparticles showed stronger brain targeting properties, which is attributed to the lipophilicity of Tween 80 to cross the BBB.

Figure 10. Fluorescence in vivo imaging in C57BL/6 mice.

Discussion

Previous reports have shown that Tween 80 can promote drug delivery through the blood–brain barrier for appropriate targeting, providing evidence for the synthesis of VCR equipped drug loaded nanoparticles [14,41,42]. Some animal studies have used the biodegradable material collagen as the drug release carrier to prepare VCR sulphate controlled release film, which can achieve the controlled release of VCR in the local lesion, increase the local drug concentration and enhance the curative effect of VCR [43]. At the same time, due to its low-dose requirements and slow-release characteristics, this method can reduce systemic side effects [44]. There were also studies in which VCR was loaded into liposomes, which can have a smaller size and a high drug loading capacity, as well as displaying slow and controlled release [15,45].

The degree of hydrophobic substitution is closely related to the properties of nanomaterials, which affects the particle size of nanoparticles. In addition, within a suitable range, the degree of substitution of the hydrophobic group is negatively correlated with the size of the nanomedicine formulation. The higher the degree of substitution is, the smaller the particle size of the nanoformulation. Therefore, we have also carried out research on the effect of the degree of hydrophobic substitution on the properties of nanomedicine. Three groups of VCR-loaded CHPT nanocomposites were designed using nanomaterials with different hydrophobic substitution degrees. In this experiment, three feeding ratios of CHP NPs were diluted to 40 nM with deionized water and loaded with Tween 80 for fluorescence detection by MST activity analysis. The Kd values of Tween 80 binding to all three researched CHPs were determined, and the results proved that CHP1 had the highest affinity for Tween 80. To explore the effect of the use of drug carriers with different hydrophobicities on the drug release rate, experimental analysis at pH 7.4 or pH 6.8 environments was conducted, and the results showed that CHPT@VCR-1 NPs had the best sustained-release control. To explore CHPT@VCR-1 NPs whether it has the best therapeutic effect, we further designed cytotoxicity experiments to compare and screen the inhibitory effects of nanoparticles on SJ-GBM2 cells. The results showed that nanoparticles have different levels of cytotoxicity, among which CHPT@VCR-1 NPs showed highest toxicity to tumour cells. After injecting VCR loaded CHPT NPs and CHP NPs into mice, nanoparticles modified with Tween 80 showed stronger fluorescence in the brain, indicating that Tween 80 adsorption on the surface of nanoparticles can increase their ability to penetrate the BBB.

In this study, an experimental scheme was designed to prepare VCR loaded CHPT nanomaterials, and the effects of different hydrophobic substitution degrees on the controllability of nanoparticle sustained-release were compared. The nanoparticles exhibited good tumour inhibition effects. Previous studies have shown that the blood–brain barrier has long been a major challenge in achieving brain targeted drug therapy [46,47]. In order to achieve brain targeting of nanoparticles and enhance their ability to cross the BBB, receptor ligand mediation is now widely used. In this study, the method of adsorbing Tween 80 can achieve good blood–brain barrier permeability. This method is simple and effective and has reference value for other studies. In future research, we will further evaluate CHPT@VCR-1 NPs through in vivo experiments.

Conclusion

The study indicated that the functionality and performance of nanoparticles largely depend on the degree of hydrophobic substitution of the polymer. Within a certain range, nanoparticles with the highest degree of hydrophobic substitution have the smallest size, the highest drug loading and the best sustained release effect. CHPT@VCR-1 NPs prepared with CHP-1 as the main body have a higher drug release rate and higher affinity for Tween 80. CHPT@VCR-1 NPs showed better cell inhibition and brain targeting. The nanomaterials in the study were loaded with drugs through physical methods, which can be used as carriers to encapsulate other drugs and exert specific therapeutic effects. In this study, the adsorption of Tween 80 on the surface of nanoparticles was used to achieve the brain targeting ability, which is a valuable method for improving the efficacy of brain delivery drugs.

Acknowledgments

The authors gratefully acknowledge the support of the Development Fund of Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was supported by the Science and Technology Innovation Project of Hunan Province [2018SK21216] and Research Program of Health Committee of Hunan Province [202106010330] to Xiangling He; Natural Science Foundation of Hunan Province of China [2021JJ40295] to Yanlan You; the National Innovation and Entrepreneurship Training Program for College Students [S202310542064] to Shiyang Fu; and Hunan Province College Student Innovation and Entrepreneurship Project [S202310542076] to Zheda Liu.