bims-novged 2022-06-19 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2022–06–19
thirteen papers selected by
the Merkel lab, Ludwig-Maximilians University

Integrin-Targeted, Short Interfering RNA Nanocomplexes for Neuroblastoma Tumor-Specific Delivery Achieve MYCN Silencing with Improved Survival.
Design and In Vitro Evaluation of Novel Cationic Lipids for siRNA Delivery in Breast Cancer Cell Lines.
Recent advances in exosome-mediated nucleic acid delivery for cancer therapy.
Recent advances in mRNA-LNP therapeutics: immunological and pharmacological aspects.
Reverse relation between cytotoxicity and Polyethylenimine/DNA ratio, the effect of using HEPES-buffered saline (HBS) medium in gene delivery.
Targeted liposomes for combined delivery of artesunate and temozolomide to resistant glioblastoma.
Spectroscopy-Based Local Modeling Method for High-Throughput Quantification of Nucleic Acid Loading in Lipid Nanoparticles.
A Biopharmaceutical Perspective on Higher-Order Structure and Thermal Stability of mRNA Vaccines.
CRISPR/Cas9 mediated somatic gene therapy for insertional mutations: the vibrator mouse model.
Synthesis and evaluation of a novel adapter lipid derivative for preparation of cyclic peptide-modified PEGylated liposomes: application of cyclic RGD peptide.
Potentialities and Challenges of mRNA Vaccine in Cancer Immunotherapy.
Cyclic Disulfide Liposomes for Membrane Functionalization and Cellular Delivery.
Fast-Forming Dissolvable Redox-Responsive Hydrogels: Exploiting the Orthogonality of Thiol-Maleimide and Thiol-Disulfide Exchange Chemistry.

Adv Funct Mater. 2021 Sep 09. 31(37): 2104843

Integrin-Targeted, Short Interfering RNA Nanocomplexes for Neuroblastoma Tumor-Specific Delivery Achieve MYCN Silencing with Improved Survival.

Aristides D Tagalakis, Vignesh Jayarajan, Ruhina Maeshima, Kin H Ho, Farhatullah Syed, Lin-Ping Wu, Ahmad M Aldossary, Mustafa M Munye, Talisa Mistry, Olumide Kayode Ogunbiyi, Arturo Sala, Joseph F Standing, Seyed M Moghimi, Andrew W Stoker, Stephen L Hart.

  The authors aim to develop siRNA therapeutics for cancer that can be administered systemically to target tumors and retard their growth. The efficacy of systemic delivery of siRNA to tumors with nanoparticles based on lipids or polymers is often compromised by their rapid clearance from the circulation by the liver. Here, multifunctional cationic and anionic siRNA nanoparticle formulations are described, termed receptor-targeted nanocomplexes (RTNs), that comprise peptides for siRNA packaging into nanoparticles and receptor-mediated cell uptake, together with lipids that confer nanoparticles with stealth properties to enhance stability in the circulation, and fusogenic properties to enhance endosomal release within the cell. Intravenous administration of RTNs in mice leads to predominant accumulation in xenograft tumors, with very little detected in the liver, lung, or spleen. Although non-targeted RTNs also enter the tumor, cell uptake appears to be RGD peptide-dependent indicating integrin-mediated uptake. RTNs with siRNA against MYCN (a member of the Myc family of transcription factors) in mice with MYCN-amplified neuroblastoma tumors show significant retardation of xenograft tumor growth and enhanced survival. This study shows that RTN formulations can achieve specific tumor-targeting, with minimal clearance by the liver and so enable delivery of tumor-targeted siRNA therapeutics.

Keywords:  MYCN; neuroblastomas; siRNA; tumors; tumor‐specific delivery

DOI:  https://doi.org/10.1002/adfm.202104843
Evid Based Complement Alternat Med. 2022 ;2022 9231641

Design and In Vitro Evaluation of Novel Cationic Lipids for siRNA Delivery in Breast Cancer Cell Lines.

Sandeep Vaidya, Manish Kumar Jeengar, Mohammad Ahmad Wadaan, Shahid Mahboob, Pankaj Kumar, Lisa M Reece, Surender Reddy Bathula, Mycal Dutta.

Breast cancer is the most common cause of cancer mortality in Western nations, with a terrible prognosis. Many studies show that siRNA plays a role in the development of tumors by acting as a tumor suppressor and apoptosis inhibitor or both. siRNAs may be used as diagnostic and prognostic biomarkers in breast cancer. Antisurvivin siRNA was chosen as a therapeutic target in breast cancer treatment because it directly targets survivin, an inhibitor of apoptosis protein, that causes cell death. However, siRNA-based treatment has significant limitations, including a lack of tissue selectivity, a lack of effective delivery mechanisms, low cellular absorption, and the possibility of systemic toxicity. To address some of these issues, we provide a siRNA delivery method based on cationic lipids. In the recent past, cationic liposomes have displayed that they offer a remarkable perspective in proficient siRNA delivery. The presence of a positive charge plays a vital role in firm extracellular siRNA binding along with active intracellular siRNA separation and low biological adversities. Consequently, the methods for developing innovative cationic lipids through rendering and utilization of appropriate positive charges would certainly be helpful for benign and effective siRNA delivery. In the current study, an effort was made to synthesize a 3,4-dimethoxyaniline lipid (DMA) to improve the effectiveness and protection of successful siRNA delivery. DMA cationic lipid successfully delivered survivin siRNA that reduced the survivin mRNA expression, indicating the possibility of utilizing siRNA therapeutics for breast cancer. It is expected that this innovative quaternary amine-based liposome can open up new avenues in the process of developing an easy and extensively used platform for siRNA delivery. Cationic lipoplexes, a potential carrier system for siRNA-based therapies in the treatment of breast cancer, were proven by our data.

DOI: https://doi.org/10.1155/2022/9231641
J Nanobiotechnology. 2022 Jun 14. 20(1): 279

Recent advances in exosome-mediated nucleic acid delivery for cancer therapy.

Ying Zhang, Qiqi Liu, Xinmeng Zhang, Haoqiang Huang, Shiqi Tang, Yujuan Chai, Zhourui Xu, Meirong Li, Xin Chen, Jia Liu, Chengbin Yang.

  Cancer is a leading public health problem worldwide. Its treatment remains a daunting challenge, although significant progress has been made in existing treatments in recent years. A large concern is the poor therapeutic effect due to lack of specificity and low bioavailability. Gene therapy has recently emerged as a powerful tool for cancer therapy. However, delivery methods limit its therapeutic effects. Exosomes, a subset of extracellular vesicles secreted by most cells, have the characteristics of good biocompatibility, low toxicity and immunogenicity, and great designability. In the past decades, as therapeutic carriers and diagnostic markers, they have caught extensive attention. This review introduced the characteristics of exosomes, and focused on their applications as delivery carriers in DNA, messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), circular RNA (circRNA) and other nucleic acids. Meanwhile, their application in cancer therapy and exosome-based clinical trials were presented and discussed. Through systematic summarization and analysis, the recent advances and current challenges of exosome-mediated nucleic acid delivery for cancer therapy are introduced, which will provide a theoretical basis for the development of nucleic acid drugs.

Keywords:  Cancer treatment; Delivery; Exosome; Gene therapy; Nucleic acid drug

DOI:  https://doi.org/10.1186/s12951-022-01472-z
J Nanobiotechnology. 2022 Jun 14. 20(1): 276

Recent advances in mRNA-LNP therapeutics: immunological and pharmacological aspects.

Seyed Hossein Kiaie, Naime Majidi Zolbanin, Armin Ahmadi, Rafieh Bagherifar, Hadi Valizadeh, Fatah Kashanchi, Reza Jafari.

  In the last decade, the development of messenger RNA (mRNA) therapeutics by lipid nanoparticles (LNP) leads to facilitate clinical trial recruitment, which improves the efficacy of treatment modality to a large extent. Although mRNA-LNP vaccine platforms for the COVID-19 pandemic demonstrated high efficiency, safety and adverse effects challenges due to the uncontrolled immune responses and inappropriate pharmacological interventions could limit this tremendous efficacy. The current study reveals the interplay of immune responses with LNP compositions and characterization and clarifies the interaction of mRNA-LNP therapeutics with dendritic, macrophages, neutrophile cells, and complement. Then, pharmacological profiles for mRNA-LNP delivery, including pharmacokinetics and cellular trafficking, were discussed in detail in cancer types and infectious diseases. This review study opens a new and vital landscape to improve multidisciplinary therapeutics on mRNA-LNP through modulation of immunopharmacological responses in clinical trials.

Keywords:  Dendritic cell; Immune system; Immunogenicity; Lipid nanoparticles; Pharmacologic response; Toll-like receptor; mRNA delivery

DOI:  https://doi.org/10.1186/s12951-022-01478-7
Toxicol In Vitro. 2022 Jun 08. pii: S0887-2333(22)00112-6. [Epub ahead of print] 105414

Reverse relation between cytotoxicity and Polyethylenimine/DNA ratio, the effect of using HEPES-buffered saline (HBS) medium in gene delivery.

Maryam Dabbaghi, Khadijeh Hashemi, Reza Kazemi Oskuee, Amir Afkhami-Goli.

  Polyethyleneimine (PEI) is considered a promising cationic polymer in non-viral gene delivery. DNA binding properties and other biochemical characteristics of PEI such as the proton sponge phenomenon, offered the branched 25 kDa PEI to be widely used for therapeutic DNA delivery, although the possible cytotoxic effects and the best conditions of PEI preparation are not still well recognized. While higher PEI/Plasmid ratios have increased transfection efficiencies, it induces more cell stress and toxicity. Considering that the PEI particle size and resulting cytotoxicity are affected by media ions, we used Neuro2A cells to assess the cell stress properties of PEI/Plasmid complexes prepared in a HEPES-buffered saline medium. Delivery of a plasmid containing EGFP happened in all increasing ratios of PEI/plasmid from 0.5, 2, 4, and 6, while higher ratios induced less unfolded protein response as evidenced by lower transcription of ER stress markers Grp78, Atf4, Chop, Xbp1, and induced Xbp1 splicing. These data were also supported by MTT cytotoxicity assay results. These findings indicate that preparing higher PEI/plasmid ratio complexes (using the equivalent of 200 ng DNA) in the HBS medium leads to less cytotoxicity.

Keywords:  Cytotoxicity; Gene delivery; HBS; Polyethyleneimine; Unfolded protein response

DOI:  https://doi.org/10.1016/j.tiv.2022.105414
Biomaterials. 2022 Jun 01. pii: S0142-9612(22)00248-4. [Epub ahead of print]287 121608

Targeted liposomes for combined delivery of artesunate and temozolomide to resistant glioblastoma.

Muhammad Ismail, Wen Yang, Yanfei Li, Tianran Chai, Dongya Zhang, Qiuli Du, Pir Muhammad, Sumaira Hanif, Meng Zheng, Bingyang Shi.

  The effective treatment of glioblastoma (GBM) is a great challenge because of the blood-brain barrier (BBB) and the growing resistance to single-agent therapeutics. Targeted combined co-delivery of drugs could circumvent these challenges; however, the absence of more effective combination drug delivery strategies presents a potent barrier. Here, a unique combination ApoE-functionalized liposomal nanoplatform based on artesunate-phosphatidylcholine (ARTPC) encapsulated with temozolomide (ApoE-ARTPC@TMZ) was presented that can successfully co-deliver dual therapeutic agents to TMZ-resistant U251-TR GBM in vivo. Examination in vitro showed ART-mediated inhibition of DNA repair through the Wnt/β-catenin signaling cascade, which also improved GBM sensitivity to TMZ, resulting in enhanced synergistic DNA damage and induction of apoptosis. In assessing BBB permeation, the targeted liposomes were able to effectively traverse the BBB through low-density lipoprotein family receptors (LDLRs)-mediated transcytosis and achieved deep intracranial tumor penetration. More importantly, the targeted combination liposomes resulted in a significant decrease of U251-TR glioma burden in vivo that, in concert, substantially improved the survival of mice. Additionally, by lowering the effective dosage of TMZ, the combination liposomes reduced systemic TMZ-induced toxicity, highlighting the preclinical potential of this novel integrative strategy to deliver combination therapies to brain tumors.

Keywords:  Artesunate-phosphatidylcholine; Liposomes; MGMT inhibition; Resistant glioblastoma; Synergistic therapy

DOI:  https://doi.org/10.1016/j.biomaterials.2022.121608
Anal Chem. 2022 Jun 14.

Spectroscopy-Based Local Modeling Method for High-Throughput Quantification of Nucleic Acid Loading in Lipid Nanoparticles.

Yuchen Fan, Zhenqi Shi, Shengli Ma, Sayyeda Zeenat Anwer Razvi, Yige Fu, Tao Chen, Jason Gruenhagen, Kelly Zhang.

Lipid nanoparticles (LNPs) are the most widely investigated delivery systems for nucleic acid-based therapeutics and vaccines. Loading efficiency of nucleic acids may vary with formulation conditions, and it is considered one of the critical quality attributes of LNP products. Current analytical methods for quantification of cargo loading in LNPs often require external standard preparations and preseparation of unloaded nucleic acids from LNPs; therefore, they are subject to tedious and lengthy procedures, LNP stability, and unpredictable recovery rates of the separated analytes. Here, we developed a modeling approach, which was based on locally weighted regression (LWR) of ultraviolet (UV) spectra of unpurified samples, to quantify the loading of nucleic acid cargos in LNPs in-situ. We trained the model to automatically tune the training library space according to the spectral features of a query sample so as to robustly predict the nucleic acid cargo concentration and rank loading capacity with similar performance as the more complicated experimental approaches. Furthermore, we successfully applied the model to a wide range of nucleic acid cargo species, including antisense oligonucleotides, single-guided RNA, and messenger RNA, in varied lipid matrices. The LWR modeling approach significantly saved analytical time and efforts by facile UV scans of 96-well sample plates within a few minutes and with minimal sample preprocessing. Our proof-of-concept study presented the very first data mining and modeling strategy to quantify nucleic acid loading in LNPs and is expected to better serve high-throughput screening workflows, thereby facilitates early-stage optimization and development of LNP formulations.

DOI: https://doi.org/10.1021/acs.analchem.2c01346
Mol Pharm. 2022 Jun 17.

A Biopharmaceutical Perspective on Higher-Order Structure and Thermal Stability of mRNA Vaccines.

Marek Kloczewiak, Jessica M Banks, Lin Jin, Mark L Brader.

  Preservation of the integrity of macromolecular higher-order structure is a tenet central to achieving biologic drug and vaccine product stability toward manufacturing, distribution, storage, handling, and administration. Given that mRNA lipid nanoparticles (mRNA-LNPs) are held together by an intricate ensemble of weak forces, there are some intriguing parallels to biologic drugs, at least at first glance. However, mRNA vaccines are not without unique formulation and stabilization challenges derived from the instability of unmodified mRNA and its limited history as a drug or vaccine. Since certain learning gained from biologic drug development may be applicable for the improvement of mRNA vaccines, we present a perspective on parallels and contrasts between the emerging role of higher-order structure pertaining to mRNA-LNPs compared to pharmaceutical proteins. In a recent publication, the location of mRNA encapsulated within lipid nanoparticles was identified, revealing new insights into the LNP structure, nanoheterogeneity, and microenvironment of the encapsulated mRNA molecules [Brader et al. Biophys. J. 2021, 120, 2766]. We extend those findings by considering the effect of encapsulation on mRNA thermal unfolding with the observation that encapsulation in LNPs increases mRNA unfolding temperatures.

Keywords:  COVID-19; RNA delivery; circular dichroism (CD); differential scanning calorimetry (DSC); lipid nanoparticle (LNP); mRNA formulation

DOI:  https://doi.org/10.1021/acs.molpharmaceut.2c00092
Precis Clin Med. 2021 Sep;4(3): 168-175

CRISPR/Cas9 mediated somatic gene therapy for insertional mutations: the vibrator mouse model.

Xin Fu, Jie Zhu, Yaou Duan, Paul Lu, Kang Zhang.

  Somatic gene therapy remains technically challenging, especially in the central nervous system (CNS). Efficiency of gene delivery, efficacy in recipient cells, and proportion of cells required for overall benefit are the key points needed to be considered in any therapeutic approach. Recent efforts have demonstrated the efficacy of RNA-guided nucleases such as CRISPR/Cas9 in correcting point mutations or removing dominant mutations. Here we used viral delivered Cas9 plasmid and two guide RNAs to remove a recessive insertional mutation, vibrator (vb), in the mouse brain. The vb mice expressed ∼20% of normal levels of phosphatidylinositol transfer protein, α (PITPα) RNA and protein due to an endogenous retrovirus inserted in intron 4, resulting in early-onset tremor, degeneration of brainstem and spinal cord neurons, and juvenile death. The in situ CRISPR/Cas9 viral treatment effectively delayed neurodegeneration, attenuated tremor, and bypassed juvenile death. Our studies demonstrate the potential of CRISPR/Cas9-mediated gene therapy for insertional mutations in the postnatal brain.

Keywords:  CRISPR/Cas9; Pitpna; neurodegenerative disease; somatic gene editing; vibrator mouse model

DOI:  https://doi.org/10.1093/pcmedi/pbab021
Eur J Pharm Sci. 2022 Jun 15. pii: S0928-0987(22)00124-5. [Epub ahead of print] 106239

Synthesis and evaluation of a novel adapter lipid derivative for preparation of cyclic peptide-modified PEGylated liposomes: application of cyclic RGD peptide.

Naoya Kato, Takumi Sato, Yuki Fuchigami, Tadaharu Suga, Geng Longjian, Masako Tsurumaru, Masayori Hagimori, Hidefumi Mukai, Shigeru Kawakami.

  Peptide ligand modified nanoparticles can simply prepared by post-insertion method to mix pre-formed nanoparticles with peptide-lipid conjugates in an aqueous solution at an optimal temperature. Therefore, water dispersibility of peptide-lipid conjugates is a very important factor for implementing the post-insertion method. We proposed that highly water dispersible peptide-lipid conjugates can be easily synthesized by separately designing novel adapter lipids with different water dispersibility and reacting them with ligands in a highly efficient manner. Adapter lipids have three critical roles; as spacers of ligand-conjugated lipids for efficient ligand presentation, as structures that form discrete molecular weight distributions, and as providing water dispersibility. In this study, we developed a novel adapter-lipid derivative that enables a variety of cyclic peptide modifications using the click reaction. The integrin αvβ3-targeted cyclic RGDfK (cRGD) peptide was selected as the cyclic peptide ligand. We designed a novel alkyne-tagged lipid with a discrete peptide spacer and bound the cRGD peptide using a click reaction to synthesize a cRGD-conjugated lipid with good water dispersibility for the preparation of cRGD-modified PEGylated liposomes using the post-insertion method. We also revealed that cRGD-modified PEGylated liposomes are efficiently associated with integrin αvβ3-expressing murine colon carcinoma (Colon-26) cells in a modification amount- and peptide sequence-dependent manner, showing high cytotoxicity upon loading with doxorubicin. This novel adapter lipid derivative can be used to synthesize various cyclic peptides by click reactions and will provide useful insights for the future development of cyclic peptide-modified PEGylated liposomes.

Keywords:  click reaction; cyclic peptides; doxorubicin delivery; liposomes; peptide-lipid conjugates

DOI:  https://doi.org/10.1016/j.ejps.2022.106239
Front Immunol. 2022 ;13 923647

Potentialities and Challenges of mRNA Vaccine in Cancer Immunotherapy.

Li-Juan Duan, Qian Wang, Cuilian Zhang, Dong-Xiao Yang, Xu-Yao Zhang.

  Immunotherapy has become the breakthrough strategies for treatment of cancer in recent years. The application of messenger RNA in cancer immunotherapy is gaining tremendous popularity as mRNA can function as an effective vector for the delivery of therapeutic antibodies on immune targets. The high efficacy, decreased toxicity, rapid manufacturing and safe administration of mRNA vaccines have great advantages over conventional vaccines. The unprecedent success of mRNA vaccines against infection has proved its effectiveness. However, the instability and inefficient delivery of mRNA has cast a shadow on the wide application of this approach. In the past decades, modifications on mRNA structure and delivery methods have been made to solve these questions. This review summarizes recent advancements of mRNA vaccines in cancer immunotherapy and the existing challenges for its clinical application, providing insights on the future optimization of mRNA vaccines for the successful treatment of cancer.

Keywords:  cancer vaccine; efficient delivery; immunotherapy; mRNA; optimization; strategies

DOI:  https://doi.org/10.3389/fimmu.2022.923647
Chemistry. 2022 Jun 14.

Cyclic Disulfide Liposomes for Membrane Functionalization and Cellular Delivery.

Megan L Qualls, Jinchao Lou, Dillon P McBee, Joshua A Baccile, Michael D Best.

  Liposomes are effective therapeutic delivery nanocarriers due to their ability to encapsulate and enhance the pharmacokinetic properties of wide-ranging therapeutics. Two primary areas in which improvement is needed for liposomal drug delivery is to enhance the ability to infiltrate cells and to easily functionalize the liposome surface. Herein, we report a liposome platform incorporating a cyclic disulfide lipid (CDL) for the dual purpose of enhancing cell entry and functionalizing the liposome membrane through thiol-disulfide exchange. In order to accomplish this, CDL-1 and CDL-2 , composed of lipoic acid (LA) or asparagusic acid (AA) appended to a lipid scaffold, were designed and synthesized. A fluorescence-based microplate immobilization assay was implemented to show that these compounds enable convenient membrane decoration through reaction with thiol-functionalized small molecules. Additionally, fluorescence microscopy experiments indicated dramatic enhancements in cellular delivery when CDLs were incorporated within liposomes. These results demonstrate that multifunctional CDLs serve as an exciting liposome system for surface decoration and enhanced cellular delivery.

Keywords:  Cellular Delivery; Cyclic Disulfide; Fluorescence Microscopy; Lipids; Liposomes

DOI:  https://doi.org/10.1002/chem.202201164
Biomacromolecules. 2022 Jun 13.

Fast-Forming Dissolvable Redox-Responsive Hydrogels: Exploiting the Orthogonality of Thiol-Maleimide and Thiol-Disulfide Exchange Chemistry.

Ismail Altinbasak, Salli Kocak, Rana Sanyal, Amitav Sanyal.

Fast-forming yet easily dissolvable hydrogels (HGs) have potential applications in wound healing, burn incidences, and delivery of therapeutic agents. Herein, a combination of a thiol-maleimide conjugation and thiol-disulfide exchange reaction is employed to fabricate fast-forming HGs which rapidly dissolve upon exposure to dithiothreitol (DTT), a nontoxic thiol-containing hydrophilic molecule. In particular, maleimide disulfide-terminated telechelic linear poly(ethylene glycol) (PEG) polymer and PEG-based tetrathiol macromonomers are employed as gel precursors, which upon mixing yield HGs within a minute. The selectivity of the thiol-maleimide conjugation in the presence of a disulfide linkage was established through 1H NMR spectroscopy and Ellman's test. Rapid degradation of HGs in the presence of thiol-containing solution was evident from the reduction in storage modulus. HGs encapsulated with fluorescent dye-labeled dextran polymers and bovine serum albumin were fabricated, and their cargo release was investigated under passive and active conditions upon exposure to DTT. One can envision that the rapid gelation and fast on-demand dissolution under relatively benign conditions would make these polymeric materials attractive for a range of biomedical applications.

DOI: https://doi.org/10.1021/acs.biomac.2c00209