Construction Of Atezolizumab Nanoimmune Micelles Loaded With As₂O₃ And Its Effect And Mechanism On Paclitaxel Resistant Cervical Cancer Cell

ObjectiveThe incidence and mortality rates of cervical cancer in China are second only to India,posing a serious threat to women’s health.In the treatment of cervical cancer,paclitaxel is a first-line chemotherapy drug.However,resistance to paclitaxel has been observed as clinical treatments progress,resulting in the failure of chemotherapy for cervical cancer.Given this challenge,there is an urgent need to identify alternative drugs specifically for cases resistant to paclitaxel.Arsenic trioxide(ATO),with the chemical formula As2O3,is a"star drug" with excellent anti-tumor efficacy.When combined with platinum-based drugs for the treatment of cervical cancer,its efficacy is comparable to the traditional combination of paclitaxel and cisplatin,both at the cellular and animal levels.Therefore,there is potential to use As2O3 as an alternative treatment regimen for cervical cancer patients resistant to paclitaxel.However,the clinical application of As2O3 in tumors urgently addresses key technical bottlenecks including:① severe systemic toxicity(obvious bone marrow suppression);②low bioavailability(hardly soluble in water,intravenous injection requiring a variety of excipients,obvious side effects);③rapid renal clearance(continuous administration for 2-4 weeks/course).Studies have confirmed that PD-L1 is highly expressed on the surface of cervical cancer cells.Therefore,it is proposed to construct As2O3-loaded anti-PD-L1 antibody nanoimmune micelles(PANDS).The structural characteristics of PANDS are as follows:①The hydrophobic core is composed of Poly(N-butyl Methacrylate)(PBMA)and Poly(N-Diisopropylaminoethyl Methacrylate)(PPDA).The carboxyl group on PBMA(-COOH)can be connected to As2O3,improving drug-loading capacity.PPDA is pH-sensitive.Under the acidic conditions of the tumor microenvironment,the amino group of PANDS can be protonated to achieve a hydrophile-lipophile balance(HLB)breaks,with the micelles depolymerize gently releasing As2O33;② The hydrophilic outer chain is composed of poly(N-acryloylmorpholine)(PNAM),promoting blood circulation time and binding to Atezolizumab,which targets PD-L1 on the surface of cervical cancer cells.PANDS is designed to achieve the following objectives:①By capitalizing on the high positivity rate of PD-L1 in cervical cancer,the targeting of As2O3 to paclitaxel-resistant cervical cancer cells can be enhanced.PANDS seeks to reduce As2O3 systemic toxicity,improve the bioavailability and blood circulation time of PANDS,and achieve sustained drug release in the tumor microenvironment.PANDS addresses the challenges associated with As2O3,including its significant systemic toxicity,low bioavailability,and rapid renal clearance.②Atezolizumab,when bound to PD-L1 on the surface of cervical cancer cells,can interrupt the PD-1/PD-L1 signaling pathway.This interaction enhances the proportion of effector immune cells in the tumor immune microenvironment,promoting the secretion of anti-tumor cytokines by the body.Consequently,PANDS amplifies the immune response against the tumor.PANDS facilitates a synergistic killing or inhibitory effect of atezolizumab and As2O3 paclitaxel-resistant cervical cancer cell strain.Methods1.The expression levels of PD-L1 on the surface of the human cervical epithelial immortalized cell H8 and cervical cancer cells HeLa,SiHa,and CaSki were measured using flow cytometry.The subcutaneous cervical cancer model in C3H/HeN mice was established by injecting them with the U14 mouse cancer cell.Tumor cells were then extracted from these mice,and the PD-L1 expression levels on the surface of U14 cells and the extracted tumor cells were measured using flow cytometry.Upon optimizing the PANDS prescription and preparation process,the hydrodynamic particle size was measured using a dynamic laser scattering instrument(DLS)and a transmission electron microscope(TEM).The in vitro safety of the nano-micelles was assessed using the CCK-8 assay.The serum stability of PANDS was verified using a bovine serum albumin(BSA)model.2.Upon establishing the paclitaxel-resistant cervical cancer cell strain(HeLa-R),the enrichment effect of PANDS on cervical cancer cells was qualitatively and quantitatively investigated using laser confocal microscopy and flow cytometry,respectively.The immunoreactivity of PANDS-linked Atezolizumab was assessed using complement dependent cytotoxicity(CDC)and antibody-dependent cell-mediated cytotoxicity(ADCC).The effects of PANDS and As2O3 on the proliferation inhibition,apoptosis induction,and invasion suppression of HeLa-R cells were compared.Transcriptomic sequencing of untreated and PANDS-treated HeLa and HeLa-R cells was carried out,followed by further bioinformatic analysis to identify potential mechanisms of PANDS on HeLa-R.Using Western Blot,the regulatory effects of PANDS on relevant pathways in HeLa-R cells were measured,and key proteins associated with paclitaxel resistance,including P-glycoprotein(P-gp),multidrug resistance protein 1(MRP1),and breast cancer resistance protein(BCRP),were examined as well.3.A subcutaneous cervical cancer model was established in immunocompetent C3H/HeN mice.The distribution of PANDS within tumor-bearing mice and its in vivo tumor-suppressive effects were evaluated using a live imaging system and fluorescence microscopy.Flow cytometry and flow cytometric bead array technology were employed to assess the distribution of immune cells within the tumor and cytokine levels in mice post-treatment respectively,which aimed to evaluate the influence of PANDS on immune cells in the tumor microenvironment and systemic cytokine effects.The safety profile of the nano-immune micelles was assessed by monitoring changes in mouse body weight,analyzing blood parameters,liver and kidney function post-treatment,and observing cellular morphological structures in major organs.By administering PANDS to mice via intravenous injection,further pharmacokinetic studies were conducted to evaluate the targeting capability,safety,and in vivo drug release characteristics of PANDS.Results1.The expression levels of PD-L1 on the surface of human cervical cancer cells,in ascending order,were as follows:H8<SiHa<HeLa<CaSki.In the tumor immune microenvironment in vivo,the mouse cervical cancer cell U14 also showed an increased expression of PD-L1.Consequently,the HeLa cell,with a medium level of PD-L1 expression,was chosen for subsequent human cervical cancer cell research.U14 was selected for subsequent research involving mouse cervical cancer cells.Utilizing atezolizumab as the external antibody for the nano-drug delivery micelles was feasible.The optimal formula for the nano-drug delivery micelles was:PBMA/PPDA/PNAM/As2O3=40:10:30:20(by weight).The preparation process was determined as follows:Rotary evaporation method was used for preparation,with DMAC chosen as the organic phase solvent.The oil-to-water ratio was 1:2,at a rotation speed of 20 rpm.The sonication time was 20 minutes,with a hydration temperature of 45℃ and hydration time of 2 hours.Following the optimal formula and preparation process,PANDS was synthesized.DLS indicated that the average particle size of PANDS was approximately 155 nm.Under TEM,PANDS exhibited a core-shell structure.Zeta potential measurements indicated PANDS carried a positive charge,favorable for penetrating the cervical cancer cell membrane.PANDS demonstrated excellent biocompatibility and serum stability,and its in vitro drug release exhibited a sustained release effect.2.In vitro cell model indicated that the self-constructed HeLa-R exhibited strong resistance to paclitaxel.PANDS demonstrated a more potent enrichment effect on HeLa-R cells compared to normal cervical cells.PANDS was capable of inhibiting the proliferation and invasion of HeLa-R cells while promoting apoptosis.The transcriptome sequencing results showed that for HeLa and HeLa-R cells,PANDS induced significantly more downregulated differentially expressed genes than upregulated differentially expressed genes;Combining GO and KEGG enrichment analysis,PANDS might exert against HeLa-R cell effects by inhibiting the PI3K/AKT signaling pathway.Western Blot analysis confirmed that PANDS not only reduced the expression levels of phosphorylated PI3K(p-PI3K)and phosphorylated AKT(p-AKT)in HeLa-R cells but also decreased the expression levels of key drug-resistance proteins,including P-gp,MRP1,and BCRP.3.In vivo animal models indicated that PANDS enriched tumor tissue,and significantly reduced the tumor burden of cervical cancer in mice.PANDS enhanced the proportion of immune effector cells such as T cells,cytotoxic T(Tc)cells,natural killer(NK)cells,natural killer T(NKT)cells,dendritic cells(DC cells),and M1-type macrophages within the tumor microenvironment.Meanwhile,PANDS reduced the proportion of M2-type macrophages and regulatory T cells(Treg cells).Consequently,PANDS promoted the secretion of anti-tumor cytokines in the body,such as TNF-α and IFN-γ.Before and after treating the mice with PANDS,there were no significant differences in mouse weight,complete blood count,liver and kidney function,or microscopic structure of major organs,suggesting good safety of the formulation.Under the same dosage conditions,compared to free As2O3,the encapsulated As2O3 in nano micelles had a noticeably extended retention time in the circulatory system,with a reduced clearance rate,achieving a certain sustained-release effect.ConclusionIn this study,we successfully develop a novel nano micellar immunocomplex,PANDS,loaded with As2O3 and Atezolizumab.PANDS demonstrates good biocompatibility and safely.PANDS effectively targets HeLa-R cells in the cervical cancer tumor microenvironment,enhancing the bioavailability and sustained-release properties of As2O3,and reducing the toxicity of As2O3 to normal tissues.PANDS exhibits a dual-action mechanism against cervical cancer:① It inhibits the PI3K/AKT signaling pathway in HeLa-R cells and decreases the expression levels of key drug resistance proteins such as P-gp,MRP1,and BCRP,thereby suppressing proliferation and invasion and promoting apoptosis;②It elevates the proportion of immune effector cells in the tumor microenvironment and boosts the secretion of anti-tumor cytokines.Consequently,PANDS enhances the anti-tumor immune response,providing a new direction and option for treating paclitaxel-resistant refractory cervical cancer.