Preparation Of A CDK4/6 Inhibitor Polypeptide-Based Nanomedicines For Triple-Negative Breast Cancer Treatment

Background:Triple negative breast cancer（TNBC）is defined as a molecular subtype lacking estrogen receptor（ER）,progesterone receptor（PR）and human epidermal growth factor receptor 2（HER2）.Accounting for 15-18%of all breast cancer（BC）incidence,TNBC is considered the most aggressive and heterogeneous BC subtype due to its characteristics such as younger age of onset,high recurrence rate,early onset of drug resistance,and higher risk of metastasis.In addition,TNBC lacks specific biomarkers and target genes,which leads to the lack of effective treatment strategies.Cyclin-dependent kinases 4 and 6（CDK4/6）are fundamental drivers of the cell cycle,which are closely related to the occurrence and development of BC.At present,the treatment of breast cancer（BC）with cyclin-dependent kinases 4 and 6 inhibitors（CDK4/6i）is largely attributed to their ability to selectively block tumor cells in the G1 phase of the cell cycle by inhibiting the level of Rb phosphorylation mediated by CDK4/6,especially for patients with hormone receptor-positive（HR+）,HER2-metastatic breast cancer（MBC）.Interestingly,it has been found that TNBC proliferation depends on CDK4/6,and CDK4/6i exerts its inhibitory effect on cell cycle from G1 to S phase by regulating Rb protein.However,TNBC have a high content of lysosomes that isolate CDK4/6i,making it difficult for drugs to be delivered to the cell cycle protein-CDK complex to exert an inhibitory effect,which is currently an urgent problem to be solved.Polypeptide-based nanoparticles delivery system can help lysosome escape,based on which we hope to realize CDK4/6i lysosome escape through polypeptide-based nanoparticles,and restore the sensitivity of TNBC to drugs.In addition,CDK4/6i nanoparticles（CDK4/6i NGs）will play a biological advantage of nanoparticles in vivo,such as improving bioavailability,promoting passive targeting of tumors,prolonging half-life in the blood,and significantly improving tumor suppression effect.CDK4/6i not only blocks the TNBC tumor cell cycle,but also regulates the tumor microenvironment（TME）to trigger an effective anti-tumor immune response.CDK4/6i reduce the number of regulatory T cells（Tregs）and enhance the function of effector T cells,thus reshape the TNBC immune microenvironment.In addition,it achieves a strong and long-lasting immunotherapy effect.Poor immune response in BC patients may be related to mechanisms such as relatively low programmed death-ligand 1（PD-L1）expression,lower neoantigen load,and insufficient tumor antigen reactivity.CDK4/6i（ABE）can enhance anti-tumor immunity by increasing the therapeutic effect of PD-L1 immune checkpoint blockade,thereby achieving a strong and lasting immune therapeutic effect.The strategy of delivering CDK4/6i by polypeptide-based nanomaterials to treat TNBC,on the one hand,inhibits the proliferation of tumors by regulating the cell cycle,and on the other hand,enhances the immune response by reshaping the immune microenvironment to play an anti-tumor role.In this study,we designed and constructed a pH and glutathione（GSH）dual-responsivepoly（ethyleneglycol）-poly(L-glutamicacid-co-L-cystine,m PEGn-P(Glu₁₀-co-Cys₅²)]nanocarrier with different PEG chain lengths to deliver CDK4/6i（ABE）overcame the difficulty of small molecule drug escape in lysosomes,remodeled the immunosuppressor microenvironment,and explored the treatment of CDK4/6i NGs combined with ICIs.This study provides a promising strategy for the treatment of TNBC with cell cycle inhibitors and helps to explore the greater potential of CDK4/6i for the treatment of TNBC.Purpose:In this study,a pH and glutathione（GSH）dual-responsive m PEGn-P(Glu₁₀-co-Cys₅²)carrier was designed to deliver CDK4/6i,and polyaminoacid CDK4/6i NGs was prepared by self-assembly.We investigated the tumor suppression effects and related mechanisms of CDK4/6 inhibitor polypeptide-based nanomedicines with different chain lengths in TNBC cells and mouse TNBC models.This study provides a theoretical basis for the development of a highly effective nanoparticle delivery system for the treatment of TNBC.Methods:（1）Synthesis and characterization of responsive CDK4/6 inhibitor nanoparticles:m PEGn-P(Glu₁₀-co-Cys₅²)nanoparticles carrier with pH and GSH dual responsiveness was synthesized by ring-opening polymerization（ROP）.The stable combination of the nanoparticle carrier and the drug is achieved through the interaction of positive and negative charges.CDK4/6 inhibitor nanoparticles（NG750/ABE,NG2000/ABE,and NG5000/ABE）were obtained via nanoprecipitation and characterized by size,zeta potential,drug loading content（DLC）,drug loading efficiency（DLE）,and responsive drug release.（2）Investigation of the lysosomal escape ability of the nanoparticles in mouse TNBC cells and the in vitro effects of CDK4/6 inhibitor nanoparticles:Flow cytometry and confocal microscopy were used to evaluate the uptake of CDK4/6 inhibitor nanoparticles by mouse TNBC cells（4T1）and their lysosomal escape ability,respectively.CCK-8 assay,flow cytometry,and fluorescence microscopy were used to evaluate the cytotoxicity,cell cycle regulation,apoptosis,and immunogenic cell death（ICD）induction of the nanoparticles.（3）To investigate the changes in metabolism and drug distribution of polypeptide-based CDK4/6i NGs in vivo,as well as the role of tumor suppression and regulation of the immune microenvironment:4T1 BC in a mouse model was established to evaluate the biological behaviors of CDK4/6 inhibitor nanoparticles.The metabolism experiment,in vivo and ex vivo imaging,tumor suppression experiment,survival experiment,flow cytometry,and ELISA were used to evaluate the effects of CDK4/6 inhibitor nanoparticles on the immune microenvironment.Immunohistochemistry was used to evaluate the expression levels of Ki-67,caspase 3,cyclin D,cyclin E and p Rb proteins in tumor tissues.Finally,the biosafety of nanodrugs was evaluated by monitoring body weight,biochemical indicators,routine blood tests,and histopathological analysis of organs.Results:（1）Nuclear magnetic resonance hydrogen spectrum,Fourier transform infrared spectroscopy,and elemental analysis confirmed the successful preparation of the material.The CDK4/6i（ABE）nanoparticle drugs NG750/ABE,NG2000/ABE,NG5000/ABE have uniform particle sizes,namely 123.1±1.4 nm,89.3±1.7 nm,and 118.8±3.8 nm respectively;the potentials are-10.6±1.8 m V,-15.7±1.3m V,-12.9±0.5 m V respectively,showing good stability.The drug loading DLC achieved by the interaction of positive and negative charges were 22.5%,18.5%,and14.7%respectively;the DLE were 81%,79%,and 37%respectively.The CDK4/6i（ABE）nanoparticle drugs have responsive structural collapse in a pH 5.5 and GSH environment,allowing for the targeted release of the ABE drug.（2）The uptake of CDK4/6i（ABE）nanoparticle drugs by murine triple-negative breast cancer cells（4T1）was significantly different and time-dependent,with Cy5.5-NG2000 having the highest cell uptake efficiency.Co-localization analysis confirmed the lysosomal escape capability of the CDK4/6i（ABE）nanoparticle drugs.The effects of the three CDK4/6i（ABE）nanoparticle drugs were superior to the free drug,and among the three nanoparticle drugs,NG2000/ABE showed stronger cell toxicity,cell cycle inhibitory effects,cell apoptosis levels,and induced the best ICD effects.（3）CDK4/6i（ABE）nanoparticle drugs can extend the blood circulation time of the drug,with the half-life of NG2000/ABE being five times that of the free drug.In vivo and ex vivo imaging showed that the CDK4/6i（ABE）nanoparticle drugs passively accumulated in TNBC,with NG2000 showing the strongest fluorescence intensity and the longest duration.NG2000/ABE showed the best tumor inhibition effect,with a tumor inhibition rate of 82.2%and the best survival extension effect.NG2000/ABE in combination withαPD-L1 has the potential to treat TNBC.NG2000/ABE has ability to regulate the immune microenvironment,increasing CD4⁺T and CD8⁺T cells,inhibiting Treg cells,enhancing anti-tumor cytokines IFN-γ,TNF-α,and IL-2,and reducing immune tolerance factor TGF-β.Immunohistochemical staining at the protein level confirmed the superior tumor suppressive effect of CDK4/6i NGs compared to free drugs.Monitoring of body weight,biochemical indicators,hemolysis experiments,routine blood tests,and histopathology of organs confirmed the reliable biosafety of nanoparticle drugs.Conclusion:（1）Three kinds of CDK4/6 inhibitor polypeptide-based nanomedicines were successfully constructed.（2）CDK4/6 inhibitor polypeptide-based nanomedicines significantly inhibited the growth of TNBC.（3）Polypeptide-based CDK4/6i NGs overcome the difficulty of lysosomal escape of free drugs in TNBC and improve the sensitivity of TNBC to drugs.（4）Polypeptide-based CDK4/6i NGs prolong the half-life of drug circulation,reshape the immune microenvironment of TNBC,with remarkable inhibitory effects on TNBC growth.