PD-L1 Monoclonal Antibody-Conjugated PEGylated Nanoparticles forBrain Cancer Immuno-Chemotherapy via Activation of STING Pathway

doi:10.21203/rs.3.rs-4982675/v1

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PD-L1 Monoclonal Antibody-Conjugated PEGylated Nanoparticles forBrain Cancer Immuno-Chemotherapy via Activation of STING Pathway

https://doi.org/10.21203/rs.3.rs-4982675/v1

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Immunotherapy for cancer is regarded as an alternative to conventional chemotherapy. Novel approach to cancer treatment called STING-targeted activation is being studied in great detail. Thus, self-assembly PEGylated coated, ovalbumin (OVA), loaded PC7A polymer, conjugated with PD-L1 (programmed death-ligand-1) monoclonal antibody (mAb) [OVA-PEG-PC7-A-mAb] was prepared by emulsion solvent evaporation. Further, physicochemical characterization and release kinetic was performed. Further, PD-L1-mAb-conjugated NPs, investigated for cellular-uptake, cytotoxic-effects, and apoptosis in PD-L1-expressing human GC cell lines, and in vivo pharmacokinetic and bio-distribution study was performed. The investigation of structure and interaction activity of PD-L1-OVA-PEG-PC7-A-mAb NPs involved the utilization of X-ray photoelectron spectroscopy, which confirmed the presence of PD-L1-mAbs on the surface of NPs. Cellular-uptake examination demonstrated that NPs conjugated with antibodies exhibited expressively enhanced levels of cellular-uptake. NPs demonstrated by in-vitro cytotoxicity experiment on 6-glioma cell lines proof the targeting efficiency. Ova-PEG-PC7-A-mAb, NPs showed greatest STING pathway activation, confirmed by higher release of IFN-𝛽) and IL-6. In-vivo study biodistribution and pharmacokinetic confirm the specific targeting in brain region. Our findings, presented here, demonstrate that biological immune-therapeutic potential of OVA-PEG-PC7-A-mAb NPs by activation mechanism of the STING pathway using PD-L1 mAbs in brain cancer therapy.

immunotherapy

nanoparticle

ovalbumine

stimulator of interferon genes (STING)

One of the worst diseases in the world today is cancer. Conventional treatment approaches consist of a mix of procedures wherein the patient is first treated with immunotherapy, radiation, or chemotherapy after the original tumour has been surgically removed [1], [2]. Even while these methods have proven to be successful, a growing number of patients are experiencing significant adverse effects from their therapies, and more concerningly, there are reports of drug-resistant tumours, relapses, and metastatic tumours. Enhancing immunotherapeutic tactics has been a focus of significant attention, as it is one of the most promising therapeutic approaches for treating difficult tumours [3]. Currently, the primary reason immunostimulating drugs have limited therapeutic efficacy is the immunosuppressive milieu. In order to get around this restriction, a lot of research has been done on drugs that have the ability to rewire the immunosuppressive microenvironment. According to recent studies, activating the stimulator of interferon genes (STING) pathway is one of the most promising methods to improve the immunotherapeutic impact [4], [5].

STING plays a critical role in innate immunity, acting as a vital defender against both infections and cancer by responding to cGAMP, a cyclic dinucleotide produced by cGAMP synthase (cGAS) upon sensing cytosolic DNA as a danger signal. This activation triggers a multifaceted type-I interferon (IFN-I) response, facilitating dendritic cell (DC) maturation and movement, while also priming cytotoxic T lymphocytes and natural killer (NK) cells for rapid immune responses [6], [7]. STING has recently gained attention as a key target for initiating anti-tumor immune pathways in cancer immunotherapy. Previous studies have observed punctate structures upon cGAMP introduction to STING, suggesting that oligomerization or higher-order architectural arrangement may be crucial for activation [8], [9]. However, therapeutic introduction of cGAMP into target cell cytosol, where STING is located, faces challenges due to its small size, doubly negative charge, swift enzymatic breakdown, rapid clearance, and potential off-target toxicity. Consequently, the pharmaceutical sector is investing significant resources into modifying natural cyclic dinucleotides (CDNs) and developing novel STING agonists to enhance their bioavailability and pharmacological efficacy. Despite promising therapeutic potential, several small-molecule STING agonists have shown limited anti-tumor efficacy and dose-dependent toxicity in early-phase clinical trials [10], [11].

Polyvalent phase condensation is essential for regulating various biological processes, including ribosome assembly, gene expression, and signal transduction, by facilitating the assembly of large molecular complexes through interactions at multiple binding sites. A previous study underscored the importance of DNA-induced liquid phase separation of cGAS in initiating innate immunity [12]. Formation of biomolecular condensates concentrates signaling pathway proteins into membraneless structures, enhancing responses to subtle cellular environment changes. These condensates exhibit transient and dynamic behavior in response to specific stimuli or stressors, varying in size from hundreds of nanometers to micrometers [13], [14].

Previous research has identified a polymer known as PC7A, distinguished by its cyclic seven-membered ring structure, which displayed potent vaccine adjuvant properties via the STING-dependent pathway [15], [16]. This study reveals PC7A's function as a polyvalent STING agonist, leveraging polymer-induced phase condensation of STING to initiate an innate immune response with sustained cytokine expression compared to cGAMP. The degree of STING activation depends on the polymer length and thus, the valency of the interaction. Furthermore, our research demonstrates that PC7A nanoparticles (NPs) loaded with cGAMP effectively inhibit tumor progression and enhance survival rates in two animal tumor models. This strategy showcases synergistic STING activation in excised human tumors and lymph nodes, providing a proof of concept for innovative cancer immunotherapy approaches targeting the STING pathway [17], [18].

PD-1, or programmed cell death protein 1, is a receptor located on the surface of cells, which serves a crucial function in dampening immune response and facilitating self-tolerance by inhibiting the inflammatory activity of T cells. Two ligands, PD-L1 and PD-L2, are bound by PD-1. An inhibitory signal is transmitted when PD-L1 binds to PD-1, which inhibits the growth of T lymphocytes that are specific to antigens in lymph nodes [19]. An effective treatment option for advanced gastric cancer (GC) involves the use of PD-L1 monoclonal antibodies (mAb). These antibodies serve as an excellent therapeutic target because they can precisely bind to extracellular domain of the PD-L1 protein, thereby inhibiting interaction between PD-1 and its ligands [20].

In this research paper, to demonstrate the viability of the antibody conjugation approach, which involved loading of ovalbumin (Ova) into PEG-PC7-A NPs after they had been modified with a PD-L1 mAb. Thus, developed an Ova-PEG-PC7-A-mAb NPs and examined their physicochemical properties, cytotoxicity, and apoptosis. Additionally, our research has revealed a significant contrast in surface density of targeted molecules between the transfected cells containing a PD-L1 expression plasmid and the untransfected control cells. This surface density is thought to be a crucial attribute in terms of cancer cell suppression efficacy. A confocal cell laser scanning microscope was used to qualitatively examine the in vitro cellular uptake of NPs. Using the MTT assay, the in-vitro cytotoxicity, in-vivo biodistribution was assessed [21]. Further, also evaluate the activation of STING pathway to check the improvement of immunotherapy and chemotherapy.

The delivery of drug is the potential to apply a prolonged drug release strategy with healthy cells while specifically delivering a higher dose of therapeutic substance to cancer cells. Ligand bind NPs appear to hold, the most promise compared to non-specific delivery devices. The effective, well-design preparation of functional nanocarriers relies heavily on choice of matrix materials used and surface functionalization [22]. The NPs are made up of a PC7-A core and a PEG-coated layer, which enables passive targeting which serves as reaction site for functional molecular decorating. A weakly soluble medicines are contained in an inner hydrophobic core of the amphiphilic NPs, which is protected from inactivation in biological settings by an outer hydrophilic shell.

Ovalbumin (Ova) was purchased from Thermo Fisher Scientific India, while PEG provided as gift sample from Evonik Industries located in Mumbai, India. Sigma Aldrich provided the PC7-A and antibody PD-L1. Analytical-grade methanol, acetone, and trifluoroacetic acid were procured from SD Fine-Chem Ltd. The C6 glioma cell line was generously provided by the National Centre for Cell Science (NCCS), located in Pune, India. Thermo Fisher Scientific - Gibco and Himedia, based in Mumbai, India, supplied the high-glucose Dulbecco's modified Eagle's medium (DMEM) and DAPI.

2.1 Detection of OVA by HPLC

Hewlitt Packard 1100 Series, Santa Clara, CA, USA) devised a universal RP-HPLC (reverse-phase high-performance liquid chromatography) method to quantify the ovalbumin (OVA). Various concentrations were used to develop a standard calibration curve for OVA. The concentration ranges used to create the Ova standard curves were 5–100 µg/mL-1. A C18 column (i.d. 150 × 4.6 mm) from Phenomenex (Macclesfield, UK) was used to run all samples at 280 nm. Solvent A (0.1% Trifluoroacetic acid in water) and solvent B (100% methanol) were employed in a twenty-minute elution gradient at a flow rate of 1 mL/min [23], [24]. The gradient was 100:0 (A:B) for the first ten minutes, 0:100 (A:B) at minute 10.1, and 100:0 (A:B) from minute 15.1 to minute 20. Graph was plotted the concentration vs time and calculate the regression value.

2.2 Preparation and Surface chemistry analysis of nanoparticles

The OVA loaded and mAb conjugated OVA-PEG-PC7-A-mAb NPs was prepared by emulsion solvent evaporation method. In this, dissolving PC7-A (1) in PEG (3 mL) solution. Then, this polymer solution was dissolve in 1.0 mL of organic solvent methanol in a beaker. This mixture of organic solvent was added dropwise to 5mL of polyvinyl alcohol (PVA, 3%w/v) solution containing ovalbumin (3mg OVA) and monoclonal antibody (mAb), followed by sonication (Misonix Inc., Farmingdale, XL2000; NY, USA) for a minute to achieve an oil-in-water (O/W) emulsion [25], [26].

Subsequently, prepared emulsion was again emulsified with an aqueous solution (5mL) containing PVA (0.5% (w/v) by sonication a minute. The O/W emulsion formed was softly stirred at room temperature in a fume hood until the organic solvent completely evaporated. The resulting solution was then filtered through a 0.45-µm microporous membrane to remove any non-incorporated drugs [27]. Blank nanoparticles (NPs) were prepared using a comparable procedure, with the exclusion of OVA addition. Fluorescent loaded NPs were produced using the same process, with Coumarin 6 replacing OVA.

2.3 Surface chemistry analysis

Presence of monoclonal antibodies (mAbs) on the nanoparticles' (NPs') surface was verified by XPS (X-ray photoelectron spectroscopy) [AXIS His-165 Ultra from Kratos Analytical (Shimadzu Corporation, Kyoto, Japan)]. Surface chemistry of the NPs was examined using XPS, with an emphasis on the elements' specific binding energies (eV) [28]. A passing energy of 80 eV was used in the fixed transmission mode, and spectrum of binding energy was obtained between 0 and 1,500 eV.

In particular, nitrogen element was investigated at 0.5 eV in specific mode. This thorough examination verified the existence of mAbs by illuminating the components' distribution and composition on the NPs' surface.

2.4 Physicochemical characterization of NPs

Size, polydispersity, zeta potential, and morphology of NPs

Using Dynamic Light Scattering (DLS), the particle size distribution in PBS buffer was evaluated. ZETAP (Electrophoretic Light Scattering) was utilised to determine OVA nanoparticles' potential. For every sample, three separate measurements were made, and the results were averaged to calculate the zeta potential, polydispersity index, and particle size [29].

At the Michigan Centre for Materials Engineering, scanning electron microscope (SEM) pictures were obtained using an FEI Nova 200 Nanolab SEM/FIB at 5kV acceleration voltages. ImageJ (Wayne Rasband, NIH) was used for image processing in order to obtain the relevant nanoparticle size distribution. Using ImageJ, measurements were made for more than 500 particles in each sample to determine the size of the particles [30].

Transmission electron microscopy (TEM): The freshly prepared NPs had been diluted with 1 mL of demineralized water and sonicated for five minutes, a little drop was placed on a circular copper grid and left to air dry. A TEM microscope (Tenai G2 20 Twin, FEI Company, Netherlands) was used to view the samples after they had been negatively stained with phosphotungstic acid (2% w/v) [31].

2.5 OVA loading and entrapment efficiency

The indirect method was applied for the determination of Ova from the OVA-PEG-PC7-A-mAb NPs. 1mL of NPs was taken in eppendorf tube and centrifuged for 15 min for 6000rpm. The supernatant was collected and proceed for the HPLC analysis as we previously described. The samples were analyzed at 280 nm using a C18 column (inner diameter 150 × 4.6 mm) with a flow rate of 1 mL/min and a twenty-minute elution gradient, composed of solvent A (0.1% trifluoroacetic acid in water) and solvent B (100% methanol). The gradient consisted of 100:0 (A:B) for the first ten minutes, followed by a transition to 0:100 (A:B) at 10.1 min, and then back to the initial gradient of 100:0 (A:B) from 15.1 to 20 min [32].

The encapsulation efficiency (EE%) and loading efficiency (LC%) were calculated using the following equations:

Encapsulation Efficiency = [(Total drug content - Free drug content) / Total drug content] × 100

Loading Efficiency = (Weight of drug in NPs / Weight of feeding drug) × 100

2.6 In-vitro release study

The release study of OVA from was performed by dialysis bag method. A dialysis bag containing OVA-PEG-PC7-A-mAb NPs (approximately 0.5 mL) with a 14-kDa molecular weight threshold was sealed and bag was placed in dissolution apparatus containing PBS with gentle shaken at 37 ± 0.1°C temperature for 240 hrs. 5mL of the release media were removed at prearranged intervals and replaced with an equal volume of new medium. Further, using the same parameters as previously mentioned, HPLC analysis was used to determine the OVA [33].

2.7 Cellular uptake of NPs by fluorescence microscopy

For cellular uptake studies and fluorescence microscope analysis, Coumarin 6 dye-loaded PEG-PC7A-mAb nanoparticles (NPs) were employed. C6 glioma cell lines were cultured in 24-well plates for one day until reaching 70% confluence. Half of the cells were transfected with a PD-1 plasmid. After 48 hours, the culture medium was replaced with a suspension of Coumarin 6-loaded mAb-conjugated NPs at a concentration of 0.125 mg/mL, and cells were incubated for either 1 or 2 hours. Following incubation, cells were washed three times with PBS, fixed with 4% cold paraformaldehyde for 20 minutes, and then washed thrice with PBS. DAPI (4′,6-diamidino-2-phenylindole, a fluorescent stain) was added. After two PBS washes, cells were observed under a confocal laser scanning microscope (CLSM; Olympus Fluoview FV1000; Olympus, Tokyo, Japan). Image analysis was conducted using ImageJ software (National Institute of Health, Bethesda, MD, USA).

2.8 In vitro cytotoxicity

The cytotoxic of NPs on C6 glioma cells were evaluated using the MTT assay. Cells were seeded at a density of 5×10^3 cells/well (0.1 mL) in 96-well plates. After 24 hours, the cells were divided into two groups, with or without PD-L1 plasmid transfection for 48 hours. Subsequently, the culture medium was replaced with prepared doses of PBS, Ovalbumin, PEG-PC7A, PEG-PC7-A-mAb, and Ova-PEG-PC7-A-mAb, and cells were incubated for an additional 48 hours. Following the treatment period, 20 µL of MTT solution (5 mg/mL) was added to each well. After an additional 4 hours of incubation, the MTT solution was removed, and dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals in the cells. Absorbance was measured at 570 nm using a microplate reader [36], [37]. Using the above formula for calculation of cell viability:

Cell viability (%) = [OD of test group/ OD of control group] = × 100%

All the reported results were obtained from three independent experiments, with each experiment being tested in triplicate for robustness and reliability. This rigorous experimental design enhances the statistical validity and reproducibility of the findings.

2.9 Apoptosis assay

The C6 glioma cells were plated at a density of 1×105 cells/well in six-well culture plates for the apoptosis experiment. For an overnight incubation period, the cells were subjected to 72 hours of treatment with blank (PBS), Ovalbumin, PEG-PC7-A, PEG-PC7-A-mAb, and Ova-PEG-PC7-A-mAb, NPs groups. As per the instructions provided in the cell apoptosis kit, cells were co-stained with Alexa Fluor 488 annexin-V (5 µL) and propidium iodide (1 µL of 100 µg/mL) and using the working solution (Thermo Fisher Scientific) per 100 µL of cell suspension for 15 minutes. Subsequently, with the help of flow cytometry [using a BD FACS Calibur instrument (BD Biosciences, San Jose, CA, USA)] stained cells were examined [38], [39]. Apoptotic cells were indicated by green fluorescence, dead cells were indicated by cells stained with both red and green fluorescence, and viable cells displayed little to no fluorescence.

2.10 Activation of STING pathway in cancer cell

To estimate the activation of STING pathway in cancer C6 glioma cells were plated at a density of 1×105 cells/well in six-well in 0.5ml RPMI 1640 medium culture plates. For an overnight incubation period, the cells were subjected to 72 hours of treatment with PBS, Ovalbumin, PEG-PC7-A, PEG-PC7-A-mAb, and Ova-PEG-PC7-A-mAb, NPs groups. Dendritic cells (DCs) obtained from mouse bone marrow were used to cultivate the treated cancer cells, cultured in RPMI 1640 media and their development was observed by flow cytometry [40]. Finally, the production of cytokines like interferon beta (IFN-𝛽) and interleukin-6 (IL-6) were calculated by an Elisa kit assay [41].

2.11 Pharmacokinetic studies

All animal experiments were conducted in accordance with guidelines and approved by the institutional ethics committee. The in-vivo animal research was conducted at the Deshpandey Lab in Bhopal. The BALB/c mice that were employed in the study were selected. The mice spent a week in hygienic polypropylene cages (subjected to a 12-hour cycle of light and dark) at 25°C and 40–70% relative humidity. Mice were separated into two groups of six, with an average weight of 25 grams apiece. In order to inject the free treatment (ovalbumin), it was dissolved in sterile water, and Ova-PEG-PC7-A-mAb, NPs was given intravenously (i.v.). Blood samples (about 0.5–1.0 ml) were taken at predefined intervals (0.083, 0.25, 1, 2, 4, 8, 12, and 24 hours) via the retro-orbital vein. After blood was extracted, it was put into ice-cold heparinized Eppendorf tubes. For fifteen minutes, the blood sample was centrifuged at 4000 rpm. Following separation, supernatant was shifted to a clean tube (1) and the plasma was kept for analysis at 20°C. The previously described HPLC method was used to quantitative determination of drug in sample [42], [43]. Plotting the plasma concentration against time curve yields the linearity equation of calibration. The pharmacokinetic analysis was carried out using WinNonlin 6.1 Professional software (Pharsight Corporation, NC, USA). Non-compartmental intravascular analysis was used to determine the pharmacokinetic parameters.

2.12 Bio-distribution study

The BALB/c mice were kept in a system of individually ventilated cages, fed a regular sterile diet, and allowed unlimited access to sterile water. The animals were kept in 25°C air-conditioned rooms with temperature control, with 12 light-dark cycles. The animals were split up into four groups, each consisting of six animals. mice were given test samples (1mg OVA/kg subsequently) such as Ovalbumin, and Ova-PEG-PC7-A-mAb, NPs. At the end of the studies, all animals were sacrificed using carbon dioxide gas after receiving their medication for a duration of 24 hours. Blood was taken in an EDTA-filled tube, and the liver, kidney, heart, lungs, brain and blood were all dissected. Tissue samples were stored at -20 C and given a single saline washing before processing [44]. The liver, kidney, heart, lung, brain, and blood were extracted, weighed, and recorded for every animal. Each organ was removed individually and placed in an oak tube before being meticulously homogenised into tiny pieces using an organ crusher (homogenizer, IKA T25 computerised Ultra-Turrax, Germany). To make tiny pieces, each organ was removed individually and placed in an oak tube before being meticulously homogenised using an organ crusher. centrifugation of the aforementioned sample (liver, kidney, heart, lungs, brain, and blood sample) at 19000 g for 15 minutes at room temperature [45]. After collecting the supernatant, an HPLC analysis was carried out.

2.13 Statistical evaluation

Every experiment was conducted in triplicate (n = 3) and the results were reported as mean ± SD. An ANOVA was used to statistically compare the results with the control group at the statistically significant level, defined as p < 0.05. All statistical analyses were performed using statistical programme (by GraphPad programme).

3.1 Preparation and Surface chemistry analysis of nanoparticles

NPs were conjugated with antibody presented in Fig. 1(A), due to amidation reaction. We also looked into the relationship between ratio of NPs to PD-L1 mAb and the quantity of antibody conjugated on NPs surface. Antibody conjugation (mAb) with NPs and surface chemistry analysis were verified by employing XPS analyze. The XPS to examine surface chemistry of NPs as well as determine the variations in the nitrogen signal corresponding to specific binding energy [46], [47]. The presence of a higher number of nitrogen atoms in the antibody molecules suggests that they should yield a stronger signal compared to the signal produced solely by amine groups in PEG-PC-7A nanoparticles. A unique peak of signals from the nitrogen orbital (N 1s) provides qualitative confirmation of the binding of antibody molecules to the polymeric matrix cores in the 'PEG-PC7A-mAb group. The polymer matrix has effectively incorporated the PD-L1 mAb, as seen by the lack of N 1s signal in the control group study (Fig. 1B).

3.2 Physicochemical characterization of NPs

The different formulations of the NPs had particle sizes and size distributions between 170 and 200 nm, with an acceptable narrow size distribution and a polydispersity of 0.203 to 0.358 as assessed by DLS (Fig. 2(A). The selected batch exhibited a size of 186.3 ± 2.5nm with a polydispersity index of 0.24 and a zeta potential of -16.15 ± 1.64. The encapsulation efficiency (EE) and loading capacity (LC) of the Ova-PEG-PC7-A-mAb nanoparticles (NPs) were determined to be 68.4%±2.5% and 61.2%±0.12%, respectively. These results indicate that the NPs have a narrow size distribution and high encapsulation efficiency. Additionally, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were employed to visualize the morphology of the Ova-PEG-PC7-A-mAb NPs. The enhanced permeability and retention (EPR) effect facilitated the accumulation of NPs in tumor tissues. Both SEM and TEM images revealed that NPs are predominantly spherical in shape, with observed particle size being consistent with that determined by dynamic light scattering (DLS) (Fig. 2B & C).

3.3 In-vitro release study

Release profile of OVA-loaded PEG-PC7-A-mAb NPs was observed in vitro for a period of ten days. In the first eight hours, OVA showed a burst release from NPs, with about 36% of the drug continuously coming out of the carriers. Subsequently, a consistent and long-lasting release was noted over the course of the next ten days, which is anticipated to furnish a drug concentration that is sustainable within an interior environment (Fig. 3 (D)). According to results findings, OVA-PEG-PC7-A-mAb NPs may be able to release OVA continuously for up to ten days.

3.4 Cellular uptake of NPs by fluorescence microscopy

Coumarin 6 fluorescence intensity in cytoplasm of PC-3 cells was measured as part of a fluorescent quantitative analysis. Under a fluorescence microscope, cellular absorption of Coumarin 6-PEG-PC7-A-mAb-NPs was examined during one hours of incubation in 6 glioma cells expressing varying amounts of PD-1 ligand expression.

The nuclei stained with DAPI are displayed in blue fluorescence in the DAPI channel; the pictures from the combined FITC and DAPI channels are displayed in column "Overlay". The photos from the FITC channel, which demonstrate the presence of Coumarin 6, are displayed in Column "Coumarin 6". The fluorescence intensity in the cytoplasm of the PD-L1 plasmid transfection group is much higher than that of the untransfected group when using the same confocal microscope and exciting laser power (Fig. 3). This result suggests that the expansion of the absorption of Coumarin 6 contained in the NPs is driven by the recognition between the PD-1 mAb on the surface of the Coumarin 6-PEG-PC7A-mAb NPs and the PD-1 ligand expressed on the cytomembrane. In spite of this, nonspecific absorption of coumarin 6 increased with time.

3.5 In vitro cytotoxicity

Cytotoxicity of NPs formulations in cancer cells is a good indicator of their capacity to cause cell death. The 6-glioma cells were classified into two groups: those that had the PD-L1 plasmid transfected and those that did not. Figure 4(A) represented the quantitative analysis of cytotoxicity of synthetic Ova-PEG-PC7-A-mAb at drug doses ranging from 20 to 150 ng/mL. It is clear that when medication concentration and PD-L1 protein expression levels rose, so did the vitality of every single cell. Both blank and Ova-unloaded NPs had very little effect on reducing cell growth throughout the range of our tested concentration.

Even at high drug concentrations prior to PD-L1 plasmid transfection, Ova-PEG-PC7-A-mAb demonstrated considerably higher cytotoxicity in the glioma cell lines as compared to control-modified NPs. The benefits become increasingly noticeable following the transfection of plasmids. In all the formulations with Ova loading, the Ova-PEG-PC7-A-mAb NPs showed the highest mortality (i.e., cytotoxicity); nevertheless, the Ova-PEG-PC7-A-mAb NPs demonstrated limited cell death at low drug dosages without PD-L1 plasmid transfection. The outcome may suggest that NPs combined with PD-L1 mAb can specifically target and cytotoxically affect cells that express the PD-L1 protein.

3.6 Apoptosis assay

After the 48 hours assessed the percentage of apoptotic cells treating with Ovalbumin, PEG-PC7-A, PEG-PC7-A-mAb, and Ova-PEG-PC7-A-mAb, NPs. The results depicted in Fig. 4(B) demonstrate that the percentage of cell death was higher when PEG-PC7-A-mAb and ovalbumin alone were incorporated into cells as opposed to the control group (16.3%±0.11% and 25.2%±0.13%, respectively). Following treatment with Ova-PEG-PC7-A-mAb NPs, there was a noticeable increase in the number of apoptotic cells (45.4%±0.23%) when compared to the control group. A substantial difference was seen when comparing the two groups that were loaded with ovalbumin and conjugated with antibodies.

3.7 Activation of STING pathway in cancer cell

STING pathway study was conducted for evaluation of NPs as therapeutic agents for enhanced immunotherapy and chemotherapy (Fig. 5(A)). Previous studies have suggested that within the STING pathway, the protein levels of phospho-tank binding kinase 1 (P-TBK1), phospho-interferon regulatory factor 3 (PIRF3), and P-STING were upregulated depending on the concentration of treatment with nanoparticles and PC7-A. These results imply that nanoparticles and PC7-A has the ability to initiate the STING pathway. The Ova-PEG-PC7-A-mAb, NPs showed the greatest STING pathway activation, suggesting that polymer backbone and encapsulated ovalbumin complexes both activated STING pathway simultaneously [48].

Subsequently, an Elisa assay was employed to evaluate the impact of C6 glioma cell treatment on the cytokines interferon beta (IFN-𝛽) and interleukin-6 (IL-6). It was discovered that Ova-PEG-PC7-A-mAb, NPs therapy increased IFN-𝛽 levels by about three times (Fig. 5 (B)) and IL-6 levels by about 35 times (Fig. 5(C)), suggesting that the STING pathway was activated and that interferons and inflammatory factors were released. The higher production of IFN-𝛽 and IL-6 levels represented the B and T cell activation.

To comprehend how these pathways affect the immune response, we cultured the treated cancer cells with mouse bone marrow-derived dendritic cells and observed their development using flow cytometry. It has been shown that the Ova, PEG-PC7-A, PEG-PC7-A and Ova-PEG-PC7-A-mAb, increase the quantity of mature dendritic cells. f exhibited the greatest immunogenic impact, according to the comparison (Fig. 5(D)). Overall, it was shown that NPs effectively activated the STING pathway by two different mechanisms: the ovalbumin complex's DNA damage and the PC7A polymer. The STING pathway's activation has been shown to encourage dendritic cell maturation, which in turn has improved the immune response [49].

3.8 Pharmacokinetic study

The relative plasma concentration-time profiles of ovalbumin and Ova-PEG-PC7-A-mAb, NPs for a period of up to 48 hours presented in Fig. 6. The pharmacokinetic parameter analysis performed by the WinNonlin 6.4 programme. The pure drug pharmacokinetic value decreased quickly up to an hour, then increased slightly at around 1.5 hours, then rapidly failed until it was no longer apparent at 2.5 hours. Conversely NPs had a sharp decrease at 2.5 hours, a peak at 10 hours, and continued declines until it was undetectable at 24 hours. The pure drug and formulation had initial plasma concentrations (C0) that were statistically analogous. The area under the curve (AUC) and plasma half-life (t_1/2) of pure drug were determined to be approximately 7.0 and 12.5 times greater, respectively, than those of the pure drug solution. This increase in AUC and t_1/2 could be attributed to the stealthy nature of the OVA present. The clearance (CL) value of OVA was found to be higher than that of pure drugs. OVA from blood were found to have a CL value that was roughly 5.0 times lower than that of pure drug solution. The relationship between the t1/2 and CL is inverse. Additionally, created formulation OVA had a greater volume of distribution (VL).

3.9 Bio-distribution study

The total drug amount in µg/gm tissue found in the liver, kidney, heart, lungs, brain, and blood sample after 24 hours. While it is possible that NPs were trapped or accumulated, they were primarily found in the brain, meaning that the mice's concentration of both drugs was high (Fig. 7). Following the injection of the produced formulation, OVA levels were reduced after 24 hours in all organs except the brain.

The goal of this study was to reconstruct NPs (PEG-PC7-A) for surface coating with PD-L1 monoclonal antibodies to form Ova-PEG-PC7-A-mAb, NPs in order to deliver anticancer drug like OVA specifically to the brain cancer. A multifunctional NPs system shows cancer treatment the as well as immunotherapy by dual activation of the STING pathway, without the use of hazardous adjuvants, minimise side effects, attain targeted drug delivery and foster synergistic therapeutic effects with cancer immune checkpoints. Targeted chemotherapy for PD-L1-positive tumours may be used with PD-L1 mAb-conjugated Ova-PEG-PC7-A-mAb NPs. In vivo tests proof the targeted delivery and measure the kinetic profile of formulation.

Acknowledgment

The authors are thankful to the Chandigarh University, for providing the necessary facilities.

The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through Large Groups [RGP. 2/66/45] and thank the Deanship of Scientific Research at Shaqra University for supporting this work.

Funding

Prof. Dr. Perwez Alam extend his appreciation to the Researchers Supporting Project number (RSPD2024R945), King Saud University, Riyadh, Saudi Arabia.

Authors’ contribution

All authors have an equal contribution.

Conflict of interest

The author declares that there is no conflict of interest in this article

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PD-L1 Monoclonal Antibody-Conjugated PEGylated Nanoparticles forBrain Cancer Immuno-Chemotherapy via Activation of STING Pathway

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Detection of OVA by HPLC

2.2 Preparation and Surface chemistry analysis of nanoparticles

2.3 Surface chemistry analysis

2.4 Physicochemical characterization of NPs

2.5 OVA loading and entrapment efficiency

2.6 In-vitro release study

2.7 Cellular uptake of NPs by fluorescence microscopy

2.8 In vitro cytotoxicity

2.9 Apoptosis assay

2.10 Activation of STING pathway in cancer cell

2.11 Pharmacokinetic studies

2.12 Bio-distribution study

2.13 Statistical evaluation

3. Result and discussion

3.1 Preparation and Surface chemistry analysis of nanoparticles

3.2 Physicochemical characterization of NPs

3.3 In-vitro release study

3.4 Cellular uptake of NPs by fluorescence microscopy

3.5 In vitro cytotoxicity

3.6 Apoptosis assay

3.7 Activation of STING pathway in cancer cell

3.8 Pharmacokinetic study

3.9 Bio-distribution study

4. Conclusion

Declarations

References

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