Prabhjeet Singh

All Publications

• Targeted polymeric nanoconjugates for BBB penetration and time specific delivery of PDL1 blocking small peptides via LRP1 targeting in melanoma brain metastases. Sharma, S., Singh, P., Chadokiya, J., Kirane, A. R. AMER ASSOC CANCER RESEARCH. 2026

  View details for DOI 10.1158/1538-7445.AM2026-6370

  View details for Web of Science ID 001734417900010

• Systemic immune reprogramming by extracranial melanoma reshapes the melanoma brain metastasis microenvironment Singh, P., Sharma, S., Chadokiya, J., Kirane, A. AMER ASSOC CANCER RESEARCH. 2026

  View details for DOI 10.1158/1538-7445.AM2026-182

  View details for Web of Science ID 001734479500029

• Predicting targeted- and immunotherapeutic response outcomes in melanoma with single-cell Raman spectroscopy and AI. bioRxiv : the preprint server for biology Chang, K., Serasanambati, M., Ogunlade, B., Hsu, H. J., Agolia, J., Stiber, A., Gu, J., Chadokiya, J., Rodriguez, G. E., Singh, P., Sharma, S., Gonçalves, A., Verma, O., Safir, F., Vu, N., Garcia, K. C., Delitto, D., Kirane, A., Dionne, J. A. 2026

  Abstract

  Identifying reliable predictors of immunotherapeutic response in melanoma remains an outstanding challenge. Existing transcriptomic and proteomic profiling methods for the tumor-immune microenvironment (TIME) are costly and may not faithfully capture modifications actively impacting tumor behavior. Here, we present a non-destructive, single-cell approach combining Raman spectroscopy and machine learning (ML) that enables rapid cell profiling and therapeutic response prediction.We analyzed single-cell Raman spectra of mouse and human melanoma cell lines alongside nine melanoma patient-derived samples with known resistance profiles to targeted and immunotherapeutic inhibitors bemcentinib, cabozantinib, dabrafenib, nivolumab, and a combination of nivolumab and relatlimab. We assessed cell phenotyping classification and treatment resistance using random forests and feature importance analysis. For patient samples, we constructed a two-stage evaluation workflow to determine clinical drug resistance through aggregated single-cell predictions and identified corresponding highly variant spectral signatures using computational methods adapted from single-cell RNA sequencing methods.In cell lines, our approach achieved >96% differentiation accuracy across tumor microenvironment cell types and induced functional phenotypes. Persistent (drug-resistant) cells formed subclusters based on genetic mutations rather than sample origin, with Raman signatures reflecting biochemical changes relevant to therapeutic pathways. For patient samples, our workflow correctly inferred resistance likelihoods for 30 of 33 clinically-relevant patient-drug combinations (91% accuracy).Single-cell Raman spectroscopy combined with machine learning offers a scalable, prognostic platform to predict therapeutic resistance likelihood, with further potential to advance clinical, multi-omic biomarker efforts for melanoma. Our approach may improve first- and second-line therapy selection assessments for precision medicine by providing rapid, non-destructive prediction of therapeutic response based on cellular spectral profiles.

  View details for DOI 10.1101/2025.05.16.654612

  View details for PubMedID 41889902

  View details for PubMedCentralID PMC13014145

• cRGD-Functionalized Nanohybrid Conjugates Codelivering Temozolomide and Rapamycin for Treating Glioblastoma Multiforme: <i>In Vitro</i> and <i>In Vivo</i> Evaluation MOLECULAR PHARMACEUTICS Singh, P., Mittal, A., Chitkara, D. 2025

  Abstract

  The conventional treatment of glioblastoma multiforme primarily uses Temozolomide as a chemoadjuvant alongside debulking surgery and radiotherapy; however, resistance, as well as tumor recurrence, remains a common outcome. Employing combination therapy to target multiple pathways while improving delivery with advanced systems has always been sought after. Herein, we report cRGD-functionalized hybrid polymeric nanoconjugates that could deliver Temozolomide (TMZ) and rapamycin (RAP) (cRGD-Hybrid TMZ/R NCs) as a dual payload. The cRGD-Hybrid TMZ/R NCs were thoroughly characterized, exhibiting a particle size and surface zeta potential of 141.83 nm (PDI 0.233) and -0.168 mV, respectively. The nanoconjugates carrying TMZ and RAP as dual payloads were initially screened for synergism by determining their combination index in C6 and U87MG glioma cells. Further, in vitro cell-based assays showed improved cellular uptake, cytotoxicity, and apoptotic potential of hybrid nanoconjugates. Thereafter, the cRGD-Hybrid TMZ/R NCs were evaluated in a C6-cell-induced syngeneic orthotropic glioma model in Sprague-Dawley rats, exhibiting an improved therapeutic outcome including, reduced hemispherical width (RH/LH) ratios, tumor burden, and improved survival rates compared to the free drug(s) counterpart. The histopathological evaluation indicated no major sign of toxicity in vital organs such as heart, lungs, liver, kidney, and spleen, affirming the biocompatibility of the developed cRGD-Hybrid TMZ/R nanoconjugates. In conclusion, amalgamating the cRGD cell-penetrating peptide with polymer hybrid nanoconjugates presents a better approach for delivering multiple payloads in the treatment of GBM.

  View details for DOI 10.1021/acs.molpharmaceut.5c00229

  View details for Web of Science ID 001599155500001

  View details for PubMedID 41128328

• Simultaneous estimation of rapamycin, temozolomide and its metabolites using UPLC-ESI-MS/MS and its application to pharmacokinetics in C6-glioma bearing animals MICROCHEMICAL JOURNAL Singh, P., Mittal, A., Chitkara, D. 2025; 216

  View details for DOI 10.1016/j.microc.2025.114567

  View details for Web of Science ID 001544241300001

• Antibody-Free Immunopeptide Nanoconjugates for Brain-Targeted Drug Delivery in Glioblastoma Multiforme. Bioconjugate chemistry Sharma, S., Lee, D., Maity, S., Singh, P., Chadokiya, J., Mohaghegh, N., Hassani, A., Kim, H., Gangarade, A., Ljubimova, J. Y., Kirane, A., Holler, E. 2025

  Abstract

  Glioblastoma Multiforme (GBM) represents a significant clinical challenge among central nervous system tumors, with a dismal mean survival rate of less than 8 months, a statistic that has remained largely unchanged for decades (National Brain Society, 2022). The specialized intricate anatomical features of the brain, notably the blood-brain barrier (BBB), pose significant challenges to effective therapeutic interventions, limiting the potential reach of modern advancements in immunotherapy to impact these types of tumors. This study introduces an innovative, actively targeted immunotherapeutic nanoconjugate (P-12/AP-2/NCs) designed to serve as an immunotherapeutic agent capable of traversing the BBB via LRP-1 receptor-mediated transcytosis. P-12/AP-2/NCs exert their immune-modulating effects by inhibiting the PD-1/PD-L1 axis through a small-sized PD-L1/PD-L2 antagonist peptide, Aurigene NP-12 (P-12). P-12/AP-2/NCs are synthesized from completely biodegradable, functionalized high molecular weight β-poly(l-malic acid) (PMLA) polymer conjugated with P-12 and Angiopep-2 (AP-2) to yield P-12/AP-2/NCs. Evaluating nanoconjugates for BBB permeability and 3D tumor model efficacy using an in vitro BBB-Transwell spheroid-based model demonstrated successful crossing of the BBB and internalization in brain 3D tumor environments. In addition, the nanoconjugate mediated T cells' cytotoxicity on 3D tumor region death in a U87 GBM 3D spheroid model. AP-2/P-12/NCs are selectively inhibited in PD1/PDL1 interaction on T cells and the tumor site, increasing inflammatory cytokine secretion and T cell proliferation. In an in vivo murine brain environment, rhodamine fluorophore-labeled AP-2/P-12/NCs displayed significantly increased accumulation in the brain during 2-6 h time intervals postinjection with a prolonged bioavailability over unconjugated peptides. AP-2/P-12/NCs demonstrated a safety profile at both low and high doses based on major organ histopathology evaluations. Our findings introduce a novel, programmable nanoconjugate platform capable of penetrating the BBB for directed delivery of small peptides and significant immune environment modulation without utilizing antibodies, offering promise for treating challenging brain diseases such as glioblastoma multiforme and beyond.

  View details for DOI 10.1021/acs.bioconjchem.5c00168

  View details for PubMedID 40601862

• Antibody-Free Immunopeptide Nano-Conjugates for Brain-Targeted Drug Delivery in Glioblastoma Multiforme. bioRxiv : the preprint server for biology Sharma, S., Lee, D., Maity, S., Singh, P., Chadokiya, J., Mohaghegh, N., Hassani, A., Kim, H., Gangarade, A., Ljubimova, J. Y., Kirane, A., Holler, E. 2025

  Abstract

  Glioblastoma Multiforme (GBM) represents a significant clinical challenge amongst central nervous system (CNS) tumors, with a dismal mean survival rate of less than 8 months, a statistic that has remained largely unchanged for decades (National Brain Society, 2022). The specialized intricate anatomical features of the brain, notably the blood-brain barrier (BBB), pose significant challenges to effective therapeutic interventions, limiting the potential reach of modern advancements in immunotherapy to impact these types of tumors. This study introduces an innovative, actively targeted immunotherapeutic nanoconjugate (P12/AP-2/NCs) designed to serve as an immunotherapeutic agent capable of traversing the BBB via LRP-1 receptor-mediated transcytosis. P12/AP-2/NCs exert its immune-modulating effects by inhibiting the PD-1/PD-L1 axis through a small-size PD-L1/PD-L2 antagonist peptide Aurigene NP-12 (P12). P12/AP-2/NCs are synthesized from completely biodegradable, functionalized high molecular weight β-poly(L-malic acid) (PMLA) polymer, conjugated with P12 and Angiopep-2 (AP2) to yield P12/AP-2/NCs. Evaluating nanoconjugates for BBB permeability and 3-D tumor model efficacy using an in vitro BBB-Transwell spheroid based model demonstrating successful crossing of the BBB and internalization in brain 3D tumor environments. In addition, the nanoconjugate mediated T cell's cytotoxicity on 3D tumor region death in a U87 GBM 3-D spheroid model. AP2/P12/NCs is selectively inhibited in PD1/PDL1 interaction on T cells and tumor site, increasing inflammatory cytokine secretion and T cell proliferation. In an in-vivo murine brain environment, rhodamine fluorophore-labeled AP2/P12/NCs displayed significantly increased accumulation in the brain during 2-6 h time intervals post-injection with a prolonged bioavailability over unconjugated peptides. AP2/P12/NCs demonstrated a safety profile at both low and high doses based on major organ histopathology evaluations. Our findings introduce a novel, programmable nanoconjugate platform capable of penetrating the BBB for directed delivery of small peptides and significant immune environment modulation without utilizing antibodies, offering promise for treating challenging brain diseases like glioblastoma multiforme and beyond.

  View details for DOI 10.1101/2025.03.07.641755

  View details for PubMedID 40161747

  View details for PubMedCentralID PMC11952356