Beyond Lipids: Exploring Advances in Polymeric Gene Delivery in the Lipid Nanoparticles Era.

The recent success of gene therapy during the COVID-19 pandemic has underscored the importance of effective and safe delivery systems. Complementing lipid-based delivery systems, polymers present a promising alternative for gene delivery. Significant advances have been made in the recent past, with multiple clinical trials progressing beyond phase I and several companies actively working on polymeric delivery systems which provides assurance that polymeric carriers can soon achieve clinical translation. The massive advantage of structural tunability and vast chemical space of polymers is being actively leveraged to mitigate shortcomings of traditional polycationic polymers and improve the translatability of delivery systems. Tailored polymeric approaches for diverse nucleic acids and for specific subcellular targets are now being designed to improve therapeutic efficacy. This review describes the recent advances in polymer design for improved gene delivery by polyplexes and covalent polymer-nucleic acid conjugates. The review also offers a brief note on novel computational techniques for improved polymer design. We conclude with an overview of the current state of polymeric gene therapies in the clinic as well as future directions on their translation to the clinic. This article is protected by copyright. All rights reserved.

Conventional and frugal methods of estimating COVID-19-related excess deaths and undercount factors.

Across the world, the officially reported number of COVID-19 deaths is likely an undercount. Establishing true mortality is key to improving data transparency and strengthening public health systems to tackle future disease outbreaks. In this study, we estimated excess deaths during the COVID-19 pandemic in the Pune region of India. Excess deaths are defined as the number of additional deaths relative to those expected from pre-COVID-19-pandemic trends. We integrated data from: (a) epidemiological modeling using pre-pandemic all-cause mortality data, (b) discrepancies between media-reported death compensation claims and official reported mortality, and (c) the "wisdom of crowds" public surveying. Our results point to an estimated 14,770 excess deaths [95% CI 9820-22,790] in Pune from March 2020 to December 2021, of which 9093 were officially counted as COVID-19 deaths. We further calculated the undercount factor-the ratio of excess deaths to officially reported COVID-19 deaths. Our results point to an estimated undercount factor of 1.6 [95% CI 1.1-2.5]. Besides providing similar conclusions about excess deaths estimates across different methods, our study demonstrates the utility of frugal methods such as the analysis of death compensation claims and the wisdom of crowds in estimating excess mortality.

Assessing Physicochemical Stability of Monoclonal Antibodies in a Simulated Subcutaneous Environment.

Monoclonal antibodies (mAbs) are being increasingly administered by the subcutaneous (SC) route compared to the traditional intravenous route. Despite the growing popularity of the subcutaneous route, our current knowledge regarding the intricate mechanistic changes happening in the formulation after injection in the subcutaneous space, as well as the in vivo stability of administered mAbs, remains quite limited. Changes in the protein environment as it transitions from a stabilized, formulated drug product in an appropriate container closure to the SC tissue environment can drastically impact the structural stability and integrity of the injected protein. Interactions of the protein with components of the extracellular matrix can lead to changes in its structure, potentially impacting both safety and efficacy. Investigating protein stability in the SC space can enable early assessment of risk and performance of subcutaneously administered proteins influencing clinical decisions and formulation development strategies. The Subcutaneous Injection Site Simulator (SCISSOR) is a novel in vitro system that mimics the subcutaneous injection site and models the events that a protein goes through as it transitions from a stabilized formulation environment to the dynamic physiological space. In this paper, we utilize the SCISSOR to probe for biophysical and chemical changes in seven mAbs post SC injection using a variety of analytical techniques. After 24 h, all mAbs demonstrated a relative decrease in conformational stability, an increase in fragmentation, and elevated acidic species. Higher order structure analysis revealed a deviation in the secondary structure from the standard and an increase in the number of unordered species. Our findings suggest an overall reduced stability of mAbs after subcutaneous administration. This reduced stability could have a potential impact on safety and efficacy. In vitro systems such as the SCISSOR combined with downstream analyses have potential to provide valuable information for assessing the suitability of lead molecules and aid in formulation design optimized for administration in the intended body compartment, thus improving chances of clinical success.

Nanotherapeutic Approaches to Treat COVID-19-Induced Pulmonary Fibrosis.

There have been significant collaborative efforts over the past three years to develop therapies against COVID-19. During this journey, there has also been a lot of focus on understanding at-risk groups of patients who either have pre-existing conditions or have developed concomitant health conditions due to the impact of COVID-19 on the immune system. There was a high incidence of COVID-19-induced pulmonary fibrosis (PF) observed in patients. PF can cause significant morbidity and long-term disability and lead to death in the long run. Additionally, being a progressive disease, PF can also impact the patient for a long time after COVID infection and affect the overall quality of life. Although current therapies are being used as the mainstay for treating PF, there is no therapy specifically for COVID-induced PF. As observed in the treatment of other diseases, nanomedicine can show significant promise in overcoming the limitations of current anti-PF therapies. In this review, we summarize the efforts reported by various groups to develop nanomedicine therapeutics to treat COVID-induced PF. These therapies can potentially offer benefits in terms of targeted drug delivery to lungs, reduced toxicity, and ease of administration. Some of the nanotherapeutic approaches may provide benefits in terms of reduced immunogenicity owing to the tailored biological composition of the carrier as per the patient needs. In this review, we discuss cellular membrane-based nanodecoys, extracellular vesicles such as exosomes, and other nanoparticle-based approaches for potential treatment of COVID-induced PF.

Advances in Lipid-Based Codelivery Systems for Cancer and Inflammatory Diseases.

Combination therapy targeting multiple therapeutic targets is a favorable strategy to achieve better therapeutic outcomes in cancer and inflammatory diseases. Codelivery is a subfield of drug delivery that aims to achieve combined delivery of diverse therapeutic cargoes within the same delivery system, thereby ensuring delivery to the same site and providing an opportunity to tailor the release kinetics as desired. Among the wide range of materials being investigated in the design of codelivery systems, lipids have stood out on account of their low toxicity, biocompatibility, and ease of formulation scale-up. This review highlights the advances of the last decade in lipid-based codelivery systems focusing on the codelivery of drug-drug, drug-nucleic acid, nucleic acid-nucleic acid, and protein therapeutic-based combinations for targeted therapy in cancer and inflammatory diseases.

Tumor Microenvironment-Responsive Polymeric iRGD and Doxorubicin Conjugates Reduce Spontaneous Lung Metastasis in an Orthotopic Breast Cancer Model.

Tremendous progress has been made in the field of nanomedicine for cancer treatment. However, most of the research to date has been focused on inhibiting primary tumor growth with comparatively less efforts directed towards managing tumor metastasis. Here, we introduce a polymeric conjugate P-DOX-iRGD that not only significantly suppressed primary tumor growth but also substantially inhibited pulmonary metastasis in an orthotopic mouse model of breast cancer. In addition, treatment with P-DOX-iRGD markedly reduced breast cancer-induced splenomegaly and liver hematopoiesis. Interestingly, contrasting results were seen for the free form and polymeric form of DOX in vitro and in vivo, which may be attributed to the enhanced permeability and retention (EPR) effect.

Modified chitosan for effective renal delivery of siRNA to treat acute kidney injury.

Acute kidney injury (AKI) is characterized by a sudden decrease in renal function and impacts growing number of people worldwide. RNA interference (RNAi) showed potential to treat diseases with no or limited conventional therapies, including AKI. Suitable carriers are needed to protect and selectively deliver RNAi to target cells to fully explore this therapeutic modality. Here, we report on the synthesis of chitosan modified with α-cyclam-p-toluic acid (C-CS) as a novel siRNA carrier for targeted delivery to injured kidneys. We demonstrate that conjugation of the α-cyclam-p-toluic acid to chitosan imparts the C-CS polymer with targeting and antagonistic properties to cells overexpressing chemokine receptor CXCR4. In contrast, the parent α-cyclam-p-toluic acid showed no such properties. Self-assembled C-CS/siRNA nanoparticles rapidly accumulate in the injured kidneys and show long retention in renal tubules. Apoptosis and metabolic and inflammatory pathways induced by p53 are important pathological mechanisms in the development of AKI. Nanoparticles with siRNA against p53 (sip53) were formulated and intravenously injected for attenuation of IRI-AKI. Due to the favorable accumulation in injured kidneys, the treatment with C-CS/sip53 decreased renal injury, extent of renal apoptosis, macrophage and neutrophil infiltration, and improved renal function. Overall, our study suggests that C-CS/siRNA nanoparticles have the potential to effectively accumulate and deliver therapeutic siRNAs to injured kidneys through CXCR4 binding, providing a novel way for AKI therapy.

Self-Assembled Alkylated Polyamine Analogs as Supramolecular Anticancer Agents.

Conformationally restrained polyamine analog PG11047 is a well-known drug candidate that modulates polyamine metabolism and inhibits cancer cell growth in a broad spectrum of cancers. Here, we report a structure-activity relationship study of the PG11047 analogs (HPGs) containing alkyl chains of varying length, while keeping the unsaturated spermine backbone unchanged. Synthesis of higher symmetrical homologues was achieved through a synthetic route with fewer steps than the previous route to PG11047. The amphiphilic HPG analogs underwent self-assembly and formed spherically shaped nanoparticles whose size increased with the hydrophobic alkyl group's increasing chain length. Assessment of the in vitro anticancer activity showed more than an eight-fold increase in the cancer cell inhibition activity of the analogs with longer alkyl chains compared to PG11047 in human colon cancer cell line HCT116, and a more than ten-fold increase in human lung cancer cell line A549. Evaluation of the inhibition of spermine oxidase (SMOX) showed no activity for PG11047, but activity was observed for its higher symmetrical homologues. Comparison with a reference SMOX inhibitor MDL72527 showed nine-fold better activity for the best performing HPG analog.

Study of Renal Accumulation of Targeted Polycations in Acute Kidney Injury.

Acute kidney injury (AKI) is a global healthcare burden characterized by rapid loss of renal function and high morbidity and mortality. Chemokine receptor CXCR4 participates in the renal infiltration of immune cells following injury and in local inflammatory enhancement. Injured renal tubule cells overexpress CXCR4, which could be used as a target for improved drug delivery in AKI. Plerixafor is a small-molecule CXCR4 antagonist that has shown beneficial effects against AKI and has been previously developed into a polymeric analog (polymeric plerixafor, PP). With the goal of gaining a better understanding of how overall charge and hydrophilicity affect renal accumulation of PP, we have synthesized PP copolymers containing hydroxyl, carboxyl, primary amine, and alkyl moieties using Michael-type addition copolymerization. All synthesized copolymers showed excellent CXCR4-binding and inhibiting ability in vitro and improved cellular uptake in hypoxia-reoxygenation stimulated mouse tubule cells. Analysis of serum protein binding revealed that polymers with hydroxyl group modification showed the least amount of protein binding. Biodistribution of the polymers was tested in a unilateral ischemia reperfusion-induced AKI mouse model. The results showed significant differences in accumulation in the injured kidneys depending on the net charge and hydrophilicity of the polymers. The findings of this study will guide the development of polymeric drug carriers for targeted delivery to injured kidneys for better AKI therapy.

Preferential siRNA delivery to injured kidneys for combination treatment of acute kidney injury.

Acute kidney injury (AKI) is characterized by a sudden loss of renal function and is associated with high morbidity and mortality. Tumor suppressor p53 and chemokine receptor CXCR4 were both implicated in the AKI pathology. Here, we report on the development and evaluation of polymeric CXCR4 antagonist (PCX) siRNA carrier for selective delivery to injured kidneys in AKI. Our results show that PCX/siRNA nanoparticles (polyplexes) provide protection against cisplatin injury to tubule cells in vitro when both CXCR4 and p53 are inhibited. The polyplexes selectively accumulate and are retained in the injured kidneys in cisplatin and bilateral ischemia reperfusion injury models of AKI. Treating AKI with the combined CXCR4 inhibition and p53 gene silencing with the PCX/sip53 polyplexes improves kidney function and decreases renal damage. Overall, our results suggest that the PCX/sip53 polyplexes have a significant potential to enhance renal accumulation in AKI and deliver therapeutic siRNA.

Gene silencing delivery systems for the treatment of pancreatic cancer: Where and what to target next?

Despite intensive research efforts and development of numerous new anticancer drugs and treatment strategies over the past decades, there has been only very limited improvement in overall patient survival and in effective treatment options for pancreatic cancer. Current chemotherapy improves survival in terms of months and death rates in pancreatic cancer patients are almost equivalent to incidence rates. It is imperative to develop new therapeutic approaches. Among them, gene silencing shows promise of effectiveness in both tumor cells and stromal cells by inhibiting tumor-promoting genes. This review summarizes potential targets for gene silencing in both pancreatic cancer cells and abundant stromal cells focusing on non-viral delivery systems for small RNAs and discusses the potential immunological implications. The review concludes with the importance of multifactorial therapy of pancreatic cancer.