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Item Can breakthroughs in dermal and transdermal macromolecule delivery surmount existing barriers and revolutionize future therapeutics?(Springer, 2025-05) Mittal, Anupama; Chitkara, DeepakThe delivery of macromolecules through dermal and transdermal routes presents both significant challenges and transformative opportunities in therapeutic applications. This review highlights the most recent advancements and innovative strategies aimed at overcoming the barriers associated with macromolecular delivery. Cutting-edge approaches such as the use of adjuvants (e.g., hyaluronic acid-based and chemical penetration enhancers), bioactive peptides with diverse functionalities, and mechanical force techniques—including iontophoresis, microneedles, and electroporation—are thoroughly explored. While various strategies have been implemented to enhance skin delivery, they often present significant challenges, particularly for macromolecules. Addressing these challenges requires integrating novel technologies and understanding the interplay between biological barriers and delivery mechanisms. Furthermore, the role of nanotechnology, through systems like nanoemulsions, polymeric nanoparticles, and transferosomes, is examined for its ability to protect macromolecules and regulate their release. Notably, this review provides unique perspectives on the interplay between these strategies and their potential to revolutionise future therapeutics. By highlighting key trends and advancements in macromolecule delivery, this review underscores the importance of innovative approaches in overcoming existing barriers and enabling efficient drug administration.Item Autophagy as a potential therapeutic target in regulating improper cellular proliferation(Frontiers Media, 2025-05) Chitkara, DeepakAutophagy is a degradative process that makes rapid turnover of old and impaired proteins and organelles possible. It is highly instigated by stress signals, like starvation, and contributes to the cell’s homeostasis. Autophagy performs a crucial function in keeping cell genomic integrity stable. Impaired autophagic flux is implicated in neurodegenerative diseases, abnormal ageing, and cancerous diseases. In diseases like cancer, autophagy performs a dualistic function; it can have both a tumor-suppressive and supportive role. Autophagy in the initial phases of tumorigenesis maintains the integrity of the genome and, if it fails, leads to cell death, thus having a tumor-suppressive role. Meanwhile, autophagy also imparts the function of the pro-survival mechanism in the latter stages of tumorigenesis and supports the cancerous cells in surviving conditions like hypoxia and increased oxidative stress. Autophagy also helps cancerous cells develop drug resistance in some cases. Thus, modulation of the autophagic mechanism is a possible therapeutic strategy in cancer therapy as its inhibition can sensitise cancer cells to anti-cancerous drugs. The promotion of autophagy, in some cases, can also safeguard cells from toxic protein aggregation and enhanced oxidative stress. Excessive autophagy can result in autophagic cell death. Autophagy also regulates several cellular processes and cell death pathways, like apoptosis. Therefore, an in-depth knowledge of the autophagy process and its regulating molecules is critically important. Pharmaceutical small molecules or cellular target modulation can help modulate the cellular autophagy process in the context of specific disease conditions.Item Simultaneous estimation of rapamycin, temozolomide and its metabolites using UPLC-ESI-MS/MS and its application to pharmacokinetics in C6-glioma bearing animals(Elsevier, 2025-09) Mittal, Anupama; Chitkara, DeepakTherapeutic drug monitoring (TDM) plays a crucial role in the optimization of drug dosage profiles and establishing an optimal balance between toxicity and its effectiveness. Developing TDM methods for simultaneous estimation of multiple molecules with diverse physiochemical properties is quite complicated and difficult to achieve. Thus, hindering the quantitative estimation for TDM and in-depth analysis from bench to clinical settings. Therefore, the current study reports a bioanalytical method for the simultaneous determination of anticancer and immunosuppressant molecules (namely rapamycin, temozolomide, and its metabolites) in a single injection using whole blood as a biological matrix.Item cRGD-functionalized nanohybrid conjugates codelivering temozolomide and rapamycin for treating Glioblastoma multiforme: in vitro and in vivo evaluation(ACS, 2025-10) Mittal, Anupama; Chitkara, DeepakThe 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.Item Lipopolymeric nanoplex-mediated CRISPR/Cas9 delivery for VEGF-A knockdown in psoriatic angiogenesis(ACS, 2025-10) Yadav, Sushil; Mittal, Anupama; Chitkara, DeepakPsoriasis is a chronic, incurable inflammatory skin disease characterized by immune cell infiltration, aberrant keratinocyte differentiation, and enhanced angiogenesis. Overexpression of the vascular endothelial growth factor-A (VEGF-A) gene promotes angiogenesis and is essential for endothelial cell migration, adhesion, and proliferation. Therefore, downregulating VEGF-A represents a promising therapeutic strategy for angiogenesis-related disorders. We investigated the application of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein complexes (sgRNA/eGFP-Cas9 RNPs) targeting VEGF-A in psoriasis. To enable efficient delivery in vitro and in vivo, we developed lipopolymeric nanoplexes (NPXs) encapsulating sgRNA/eGFP-Cas9 RNPs. These NPXs exhibited a particle size of 142.2 nm (polydispersity index: 0.144), a zeta potential of +4.27 mV, and achieved >70% transfection efficiency in HaCaT (human immortalized keratinocyte) cells. Ex vivo skin permeation studies demonstrated 66% of permeation after 24 h. The optimized NPX formulation was incorporated into a Carbopol-based gel, which displayed non-Newtonian, shear-thinning behavior with variable thixotropy and achieved 48% of skin permeation after 24 h. In vivo efficacy assessment in an imiquimod-induced psoriasis model in Swiss albino mice showed significantly improved Psoriasis Area and Severity Index (PASI) scores, reduced epidermal damage, and suppressed keratinocyte proliferation compared to naked RNPs and blank gel controls. Gene editing analysis revealed an indel frequency of 40.7% by T7 endonuclease I assay and 14% by Sanger sequencing. Enhanced cellular uptake, efficient skin permeation and retention, and improved therapeutic efficacy collectively highlight the potential of NPX-mediated CRISPR/Cas9 delivery as a noninvasive strategy for psoriasis treatment.Item Lipo-polymeric nano-complexes for dermal delivery of a model protein(RSC, 2024-05) Chitkara, DeepakDelivering macromolecules across the skin poses challenges due to the barrier properties of stratum corneum. Different strategies have been reported to cross this barrier, such as chemical penetration enhancers and physical methods like microneedles, sonophoresis, electroporation, laser ablation, etc. Herein, we explored a cationic lipo-polymeric nanocarrier to deliver a model protein across the skin. A cationic amphiphilic lipo-polymer was used to prepare blank nanoplexes, which were subsequently complexed with anionic fluorescein-tagged bovine serum albumin (FITC-BSA). Blank nanoplexes and FITC-BSA complexed nanoplexes showed sizes of 93.72 ± 5.8 (PDI-0.250) and 145.9 ± 3.2 nm (PDI-0.258), respectively, and zeta potentials of 25.6 ± 7.0 mV and 9.17 ± 1.20 mV. In vitro cell culture, and toxicity studies showed optimal use of these nanocarriers, with hemocompatibility data indicating non-toxicity. Ex vivo skin permeation analysis showed a skin permeation rate of 33% after 24 h. The optimized formulation was loaded in a carbopol-based gel that exhibits non-Newtonian flow characteristics with shear-thinning behavior and variable thixotropy. The nanoplexes delivered via gel demonstrated skin permeation of 57% after 24 h in mice skin ex vivo. In vivo skin toxicity testing confirmed the low toxicity profile of these nanocarriers. These results are promising for the transdermal/dermal delivery of macromolecules, such as protein therapeutics, using nanoplexesItem Restoring physiological parameters of the pancreas and kidney through treatment with a polymeric nano-formulation of C-peptide and lisofylline combination in diabetic nephropathy(RSC, 2024) Chitkara, DeepakDiabetic nephropathy (DN) is a progressive kidney disorder that develops as a complication of diabetes due to long-term exposure to elevated blood glucose levels (BGLs). In this case, an intervention of therapeutic moieties is needed to target the specific elements involved in diabetes to prevent/delay the deterioration of kidney function. Therefore, the present study focused on designing and evaluating a potent nano-formulation of a combination of C-peptide (CPep) and the anti-diabetic drug lisofylline (LSF) to prevent streptozotocin (STZ)-induced DN. As a strategic intervention, an LSF-oleic acid prodrug (LSF-OA) was initially synthesized and further encapsulated in an in-house-synthesized cationic polymer [(mPEG-b-P(CB-{g-DMDP}-co-LA)); mPLM] to prepare polymeric nano-complexes of CPep via electrostatic interaction, possessing a size of 218.6 ± 14.4 nm and zeta potential of +5.2 mV together with stability for 30 days at 25 °C. mPLM-LSF-OA-CPep nanoparticles demonstrated hemocompatibility with RBCs and exhibited potent anti-oxidant activity by reducing nitrite levels, inducing the release of anti-oxidant GSH and protecting metabolically stressed rat kidneys and murine insulinoma cells from apoptosis. In vivo pharmacokinetics depicted an increase in t½ and mean residence time in rats, which further improved the BGL and renal conditions and reduced plasma IL-6 and TNF-α levels in the STZ-induced DN animal model when treated with mPLM-LSF-OA-CPep compared to free LSF and CPep. Moreover, an increase in the plasma insulin level and detection of proliferative marker cells in pancreatic islets suggested the regeneration of β-cells in diabetic animals.Item Next-generation CRISPR/Cas-based ultrasensitive diagnostic tools: current progress and prospects(RSC, 2024) Chitkara, DeepakCRISPR/Cas has been explored as a powerful molecular scissor that uses a double-strand break mediated non-homologous end joining (NHEJ) or homology-directed repair (HDR) to achieve precise gene editing. Cas effectors come in several different forms, each with its own set of features and applications. SpCas9 was the first and most extensively studied CRISPR/Cas version, and it has been hailed as a biotechnology breakthrough that could potentially correct mutations to treat genetic diseases. Recently, the Cas12 and Cas13 effector variants of Class II, Type V and Type VI, have been explored for their specific collateral cleavage (trans-cleavage) activity on target recognition. This trans-cleavage activity helps in the recognition of target nucleic acids. CRISPR diagnostics technology utilized the binding of crRNA with Cas12/13 protein to form the Ribonucleoproteins (RNPs) complex, which further cleaves the target sequence in cis-cleavage, followed by the activation of trans-cleavage of a nonspecific fluorescent DNA/RNA probe, resulting in the production of a fluorescent signal that could be quantitatively recorded. Later, nanotechnology and mobile-based detection applications were incorporated into the system to develop advanced lateral flow-based strips and are also associated with the technology to make it more feasible. Overall, this review compiles the experimental evidence consolidating the application of CRISPR/Cas as next-generation biosensors for diagnostic applications.Item Neurotensin conjugated polymeric porous microparticles suppress inflammation and improve angiogenesis aiding in diabetic wound healing(Wiley, 2024-11) Chitkara, Deepak; Mittal, AnupamaNeurotensin (NT), a bioactive tridecapeptide aids in diabetic wound healing by modulating inflammation and angiogenesis. However, its rapid degradation in peptidase-rich wound environment (plasma half-life <2 min) limits its efficacy. To address this, neurotensin-conjugated polymeric porous microparticles (NT-PMP) were developed and loaded in gelatin (hydrogel 15% w/v) for topical application, enabling sustained NT release to enhance therapeutic outcomes. NT-PMP exhibited a size range of 60 – 240 µm (mean: 120.63 ± 40.71 µm) and pore size of 5 – 16 µm (average: 10.68 ± 3.47 µm). In vitro studies demonstrated cytocompatibility of NT-PMP in fibroblasts and reduced TNF-α levels in inflammation-induced macrophages (1256 ± 167.02 pg/ml). Further NT-PMP scaffold depicted excellent cell adhesion and migration properties upon seeding of dermal fibroblasts on surface of PMPs. In vivo studies in diabetic wound rat model demonstrated effective wound management, characterized by notable regenerative and healing attributes in the presence of NT-PMP. This included complete re-epithelialization, reducing pro-inflammatory cytokine (TNF-α), and enhancing VEGF expression, ultimately leading to the development of a well-organized collagen matrix in diabetic wounds upon application of NT-PMP gel.Altogether, NT conjugated PMP loaded in hydrogel demonstrated significant regenerative and healing properties, suggesting its potential as an alternative treatment for diabetic wounds.Item Macrophage derived Exosomal Docetaxel (Exo-DTX) for pro-metastasis suppression: QbD driven formulation development, validation, in-vitro and pharmacokinetic investigation(Elsevier, 2024-02) Chitkara, DeepakExosomes, biogenic nano-vesicles, are renowned for their ability to encapsulate diverse payloads, however the systematic development and validation of exosomal formulation with significant biological implications have been overlooked. Herein, we developed and validated Exo-DTX, a QbD-driven optimized RAW 264.7 cell derived exosomal anti-cancer formulation of docetaxel (DTX) and evaluate its anti-metastatic and apoptotic efficacy in TNBC 4T1 cells. RAW264.7-derived exosomes were having particle size (112.5 ± 21.48 nm) and zeta-potential (−10.268 ± 3.66 mV) with polydispersity (PDI:0.256 ± 0.03). The statistical optimization of exosomes (200 μg) with Exo: DTX ratio 4:1 confirmed encapsulation of 23.60 ± 1.54 ng DTX/ µg exosomes. Exo-DTX (∼189 nm, −11.03 mV) with 100 ng/ml DTX as payload exhibited ∼5 folds’ improvement in IC50 of DTX and distinct cytoskeletal deformation in TNBC 4T1 cells. It also has shown enormous Filamentous actin (F-actin) degradation and triggered apoptosis explained Exo-DTX's effective anti-migratory impact with just 2.6 ± 6.33 % wound closure and 4.56 ± 1.38 % invasion. The western blot confirmed that Exo-DTX downregulated migratory protein EGFR and β1-integrin but raised cleaved caspase 3/caspase 3 (CC3/C3) ratio and BAX/BCL-2 ratio by about 2.70 and 4.04 folds respectively. The naive RAW 264.7 exosomes also contributed positively towards the effect of Exo-DTX formulation by suppressing β1-integrin expression and increasing the CC3/C3 ratio in TNBC 4T1 cells as well. Additionally, significant improvement in PK parameters of Exo-DTX was observed in comparison to Taxotere, 6-folds and 3.04-folds improved t1/2 and Vd, proving the translational value of Exo-DTX formulation. Thus, the Exo-DTX so formulated proved beneficial in controlling the aggressiveness of TNBC wherein, naive exosomes also demonstrated beneficial synergistic anti-proliferative effect in 4T1.