bims-novged 2022-09-04 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2022–09–04
eleven papers selected by
the Merkel lab, Ludwig-Maximilians University

Polyester materials for mRNA delivery.
Cancerous pH-responsive Polycarboxybetaine-coated Lipid Nanoparticle for Smart Delivery of siRNA against Subcutaneous Tumor Model in Mice.
Co-delivery of curcumin and Bcl-2 siRNA to enhance therapeutic effect against breast cancer cells using PEI-functionalized PLGA nanoparticles.
Delivery of self-replicating messenger RNA into the brain for the treatment of ischemic stroke.
Evaluation of adsorption of DNA/PEI polyplexes to tubing materials.
Engineering Lipid Nanoparticles for Enhanced Intracellular Delivery of mRNA through Inhalation.
mRNA lipid nanoparticle phase transition.
A rapid and quantitative reversed-phase HPLC-DAD/ELSD method for lipids involved in nanoparticle formulations.
Different Influences of Biotinylation and PEGylation on Cationic and Anionic Proteins for Spheroid Penetration and Intracellular Uptake to Cancer Cells.
Synthesis of doxorubicin-loaded peptosomes hybridized with gold nanorod for targeted drug delivery and CT imaging of metastatic breast cancer.
QbD-based rivastigmine tartrate-loaded solid lipid nanoparticles for enhanced intranasal delivery to the brain for Alzheimer's therapeutics.

Explor Target Antitumor Ther. 2022 ;3(2): 117-127

Polyester materials for mRNA delivery.

Wang Chen, Yonghui Ma, Xiaoxuan Liu, Dandan Zhu.

  Messenger RNA (mRNA) has recently made important progress in clinical implementation, offering a promising therapeutic option for infectious disease and cancer. However, the nature of mRNA molecules rendered them poorly bioavailable and unstable in vivo, impeding their further clinical application. Therefore, safe and efficient delivery of mRNA therapeutics to the target site is crucial for their successful translation into the clinical setting. Various vectors have been explored for mRNA delivery. Among them, polyesters and their analogs, a family of biodegradable polymers, have exhibited great potential for mRNA delivery. In this short review, the authors briefly introduce mRNA therapeutics, their therapeutic applications and delivery challenges. The authors then presented the typical examples of polyester materials for mRNA delivery to highlight the current progress and discuss the challenges for the rational design of polyester based mRNA delivery vectors. The authors hope to provide a new insight for the design of biodegradable vectors for nucleic acids delivery, thereby promoting their further clinic translation.

Keywords:  gene therapy; mRNA; mRNA delivery; poly(β-amino ester); polyester

DOI:  https://doi.org/10.37349/etat.2022.00075
Cancer Sci. 2022 Sep 01.

Cancerous pH-responsive Polycarboxybetaine-coated Lipid Nanoparticle for Smart Delivery of siRNA against Subcutaneous Tumor Model in Mice.

Yi-Jung Sung, Guo Haochen, Aria Ghasemizadeh, Xin Shen, Wanphiwat Chintrakulchai, Motoaki Kobayashi, Masahiro Toyoda, Koichi Ogi, Junya Michinishi, Tomoyuki Ohtake, Makoto Matsui, Yuto Honda, Takahiro Nomoto, Hiroyasu Takemoto, Yutaka Miura, Nobuhiro Nishiyama.

  Lipid nanoparticles (LNPs) have been commonly used as a vehicle of nucleic acids such as small interfering RNA (siRNA), and the surface modification of LNPs is one of the determinants on their delivery efficiency especially in the systemic administration. However, the applications of siRNA encapsulated LNPs are limited due to lacking effective systems to deliver to solid tumors. Here, we report a smart surface modification employing a charge-switchable ethylenediamine-based polycarboxybetaine for enhancing tumor accumulation via interaction with anionic tumorous tissue-constituents owing to selective switch to cationic charge in response to cancerous acidic pH. Our polycarboxybetaine-modified LNP could enhance cellular uptake in cancerous pH, resulting in facilitated endosomal escape and gene knockdown efficiency. After systemic administration, the polycarboxybetaine-modified LNP accomplished high tumor accumulation in SKOV3-luc and CT 26 subcutaneous tumor models. The siPLK-1-encapsulated LNP thereby accomplished significant tumor growth inhibition. This study demonstrates a promising potential of the pH-responsive polycarboxybetaine as the material modifying the surface of LNPs for efficient nucleic acid delivery.

Keywords:  Drug delivery systems; Lipid nanoparticle; Polyzwitterion; pH-responsiveness; siRNA

DOI:  https://doi.org/10.1111/cas.15554
Pharm Dev Technol. 2022 Aug 31. 1-13

Co-delivery of curcumin and Bcl-2 siRNA to enhance therapeutic effect against breast cancer cells using PEI-functionalized PLGA nanoparticles.

Leila Mohammad Gholinia Sarpoli, Shohreh Zare-Karizi, Erfan Heidari, Akbar Hasanzadeh, Mehrdad Bayandori, Fereshteh Azedi, Michael R Hamblin, Mahdi Karimi.

   PURPOSE: Breast cancer is the second major cause of death worldwide among women. Co-delivery of anticancer drugs and nucleic acids targeting the apoptosis pathway could be a promising new approach.
METHODS: In the present study, we synthesized a novel nanostructure for co-delivery of curcumin and siRNA to breast cancer cells. Curcumin-loaded polylactic-co-glycolic acid (PLGA) was synthesized using an O/W emulsion-solvent diffusion method. It was coated with polyethylenimine (PEI) and subsequently complexed with Bcl-2 siRNA. Also nanoparticles were characterized such as zeta potential, size distribution and drug encapsulation. Finally the cytotoxicity of NP and Bcl2 experession was evaluated.
RESULTS: The curcumin loaded PLGA nanoparticles were 70nm in size, and increased to 84 nm after incorporation of PEI plus Bcl-2 siRNA. The encapsulation ratio of drug in our nanoparticle was 78%. Cellular internalization PLGA-CUR-PEI/Bcl-2 siRNA NPs was confirmed by fluorescence microscopy with broadcasting of the fluorescence in the cytoplasm and into the nucleus. The results of the cell viability assay revealed that curcumin-loaded PLGA coated with PEI and Bcl-2 siRNA exhibited the highest cytotoxicity against the T47D cell line, while the siRNA decreased the Bcl-2 expression by 90.7%.
CONCLUSION: The co-delivery of curcumin plus Bcl-2 siRNA with the PLGA-PEI nanosystem could be a synergistic drug carrier against breast cancer cells.

Keywords:  Bcl-2 siRNA; Curcumin; breast cancer; nanodelivery; polylactic-co-glycolic acid

DOI:  https://doi.org/10.1080/10837450.2022.2120003
J Control Release. 2022 Aug 27. pii: S0168-3659(22)00556-9. [Epub ahead of print]

Delivery of self-replicating messenger RNA into the brain for the treatment of ischemic stroke.

Minkyung Kim, Jungju Oh, Youngki Lee, Eun-Hye Lee, Seung Hwan Ko, Ji Hoon Jeong, Chang Hwan Park, Minhyung Lee.

  Ischemic stroke is caused by the occlusion of cerebral arteries. In the ischemic stroke, ischemia-reperfusion injury increases the damage in the brain after reperfusion. In the previous study, heme oxygenase-1 (HO1) mRNA was delivered into the ischemic brain, showing that HO1-mRNA had higher therapeutic effect and less side-effect than HO1-plasmid (pHO1). However, mRNA is degraded faster than plasmid DNA reducing the duration of gene expression. In this study, self-replicating mRNA (Rep-mRNA) was developed using a replicon system from Venezuelan Equine Encephalitis virus to compensate this disadvantage of mRNA delivery. Deoxycholic acid-conjugated polyethylenimine (DA-PEI) was used as a carrier of the mRNA. The Rep-mRNA/DA-PEI complex had a size of around 90 nm and a zeta-potential of 33 mV. In the in vitro transfection assays, gene expression by the HO1-Rep-mRNA/DA-PEI complex persisted at least 14 days, while that by the HO1-mRNA/DA-PEI complex approached basal level at 3 days after transfection. Therapeutic effects of the HO1-Rep-mRNA/DA-PEI complexes were evaluated in the ischemic stroke animal models. The complexes were injected into the brain stereotaxically. HO1 expression by the HO1-Rep-mRNA/DA-PEI complex persisted at least 7 days after injection, but the pHO1/DA-PEI or HO1-mRNA/DA-PEI complex showed basal level of HO1-expression at 7 days after injection. Due to higher and longer expression of HO1, the apoptosis level and infarct size were decreased by the HO1-Rep-mRNA/DA-PEI complexes, compared with the pHO1/DA-PEI and HO1-mRNA/DA-PEI complex. These results suggest that HO1-Rep-mRNA/DA-PEI complex may have a potential as a long-lasting therapeutic system for the treatment of ischemic stroke.

Keywords:  Gene delivery; Gene therapy; Ischemia-reperfusion; Ischemic stroke; Self-replicating mRNA

DOI:  https://doi.org/10.1016/j.jconrel.2022.08.049
Eur J Pharm Biopharm. 2022 Aug 27. pii: S0939-6411(22)00180-1. [Epub ahead of print]

Evaluation of adsorption of DNA/PEI polyplexes to tubing materials.

Tobias Wm Keil, Natalie Deiringer, Wolfgang Friess, Olivia M Merkel.

  Nucleic acid drugs hold great promise for potential treatment of a variety of diseases. But efficient delivery is still the major challenge impeding translation. Nanoformulations based on polymers and lipids require preparation processes such as microfluidic mixing, spray drying or final filling, where pumping is a crucial step. Here, we studied the effect of pumping on the com-ponent and overall loss of a binary polyplex formulation made of DNA and polyethyleneimine (PEI). We varied tubing length and material with a focus on subsequent spray drying. Interestingly, product loss increased with the length of silicon tubing. Losses of DNA were prevented by using Pumpsil. The following spray drying process did not affect DNA content but caused PEI loss. Characterization of the different tubing materials revealed similar hydrophobicity of all tubing materials and showed neutral Pumpsil surface charge, negative Santoprene surface charge, and a positive Silicon surface charge. Hence, adsorption of DNA onto tubing material was concluded to be the root cause for DNA loss after pumping and is based upon an interplay of ionic and hydrophobic interactions between polyplexes and tubing material. Overall, selecting the ap-propriate tubing material for processing nucleic acid nanoparticles is key to achieving satisfactory product quality.

Keywords:  Nucleic acid therapeutics; polyplex; pumping; surface adsorption; tubing material

DOI:  https://doi.org/10.1016/j.ejpb.2022.08.014
ACS Nano. 2022 Aug 29.

Engineering Lipid Nanoparticles for Enhanced Intracellular Delivery of mRNA through Inhalation.

Jeonghwan Kim, Antony Jozic, Yuxin Lin, Yulia Eygeris, Elissa Bloom, Xiaochen Tan, Christopher Acosta, Kelvin D MacDonald, Kevin D Welsher, Gaurav Sahay.

  Despite lipid nanoparticles' (LNPs) success in the effective and safe delivery of mRNA vaccines, an inhalation-based mRNA therapy for lung diseases remains challenging. LNPs tend to disintegrate due to shear stress during aerosolization, leading to ineffective delivery. Therefore, LNPs need to remain stable through the process of nebulization and mucus penetration, yet labile enough for endosomal escape. To meet these opposing needs, we utilized PEG lipid to enhance the surficial stability of LNPs with the inclusion of a cholesterol analog, β-sitosterol, to improve endosomal escape. Increased PEG concentrations in LNPs enhanced the shear resistance and mucus penetration, while β-sitosterol provided LNPs with a polyhedral shape, facilitating endosomal escape. The optimized LNPs exhibited a uniform particle distribution, a polyhedral morphology, and a rapid mucosal diffusion with enhanced gene transfection. Inhaled LNPs led to localized protein production in the mouse lung without pulmonary or systemic toxicity. Repeated administration of these LNPs led to sustained protein production in the lungs. Lastly, mRNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR) was delivered after nebulization to a CFTR-deficient animal model, resulting in the pulmonary expression of this therapeutic protein. This study demonstrated the rational design approach for clinical translation of inhalable LNP-based mRNA therapies.

Keywords:  cystic fibrosis; inhalation; lung delivery; mRNA therapy; nebulization; pulmonary delivery; β-sitosterol

DOI:  https://doi.org/10.1021/acsnano.2c05647
Biophys J. 2022 Aug 30. pii: S0006-3495(22)00697-X. [Epub ahead of print]

mRNA lipid nanoparticle phase transition.

Marius F W Trollmann, Rainer A Böckmann.

Crucial for mRNA-based vaccines are the composition, structure and properties of lipid nanoparticles (LNPs) as their delivery vehicle. Using all-atom molecular dynamics simulations as a computational microscope, we provide an atomistic view on the structure of the Comirnaty vaccine LNP, its molecular organization, physico-chemical properties and insight in its pH-driven phase transition enabling mRNA release at atomistic resolution. At physiological pH, our simulations suggest an oil-like LNP core that is composed of the aminolipid ALC-0315 and cholesterol (ratio 72:28). It is surrounded by a lipid monolayer formed by distearoylphosphatidylcholine, ALC-0315, PEGylated lipids and cholesterol at a ratio of 22:9:6:63. Protonated aminolipids enveloping mRNA formed inverted micellar structures that provide a shielding and likely protection from environmental factors. In contrast, at low pH, the Comirnaty lipid composition instead spontaneously formed lipid bilayers that display a high degree of elasticity. These pH-dependent lipid phases suggest that a change in pH of the environment upon LNP transfer to the endosome likely acts as trigger for cargo release from the LNP core by turning aminolipids inside out thereby destabilizing both the LNP shell and the endosomal membrane.

DOI: https://doi.org/10.1016/j.bpj.2022.08.037
J Pharm Biomed Anal. 2022 Aug 19. pii: S0731-7085(22)00432-0. [Epub ahead of print]220 115011

A rapid and quantitative reversed-phase HPLC-DAD/ELSD method for lipids involved in nanoparticle formulations.

Yannick Mousli, Mathilde Brachet, Jeanne Leblond Chain, Ludivine Ferey.

  Lipid nanoparticles (LNPs) have shown great success as drug delivery systems, especially for mRNA vaccines, as those developed during the Covid-19 pandemics. Lipid analysis is critical to monitor the formulation process and control the quality of LNPs. The present study is focused on the development and validation of a high-performance liquid chromatography - diode array detector -evaporative light scattering detector (HPLC-DAD/ELSD) based method for the simultaneous quantification of 7 lipids, illustrating the main components of LNPs: ionizable lipids, the neutral co-lipid cholesterol, phospholipids, hydrophilic polymer-lipids for colloidal stability (e.g., a PEGylated lipid). In particular, this study focuses on two innovative synthetic lipids: a switchable cationic lipid (CSL3) which has demonstrated in vitro and in vivo siRNA transfection abilities, and the palmitic acid-grafted-poly(ethyloxazoline)5000 (PolyEtOx), used as an alternative polymer to address allergic reactions attributed to PEGylated lipids. The HPLC separation was achieved on a Poroshell C18 column at 50 °C using a step gradient of a mobile phase composed of water/methanol mixtures with 0.1% (v/v) trifluoroacetic acid (TFA). This method was validated following ICH Q2(R1) & (R2) guidelines in terms of linearity (R² ≥ 0.997), precision (relative standard deviation on peak areas < 5% for intermediate repeatability), accuracy (recoveries between 92.9% and 108.5%), and sensitivity. Indeed, low detection and quantitation limits were determined (between 0.02 and 0.04 µg and between 0.04 and 0.10 µg, respectively). Due to its high selectivity, this method allowed the analysis of lipid degradation products produced through degradation studies in basic, acidic, and oxidative conditions. Moreover, the method was successfully applied to the analysis of several liposome formulations at two key steps of the development process. Consequently, the reported HPLC method offers fast, versatile, selective and quantitative analysis of lipids, essential for development optimization, chemical characterization, and stability testing of LNP formulations.

Keywords:  Evaporative light scattering detection; Lipid analysis; Lipid nanoparticles; Reversed-phase HPLC; Stability study; Validation

DOI:  https://doi.org/10.1016/j.jpba.2022.115011
J Microbiol Biotechnol. 2022 Aug 24. 32(9): 1-10

Different Influences of Biotinylation and PEGylation on Cationic and Anionic Proteins for Spheroid Penetration and Intracellular Uptake to Cancer Cells.

Won Ho Jung, Gayeon You, Hyejung Mok.

  To better understand the effects of PEGylation and biotinylation on the delivery efficiency of proteins, the cationic protein lysozyme (LZ) and anionic protein bovine serum albumin (BSA) were chemically conjugated with poly(ethylene glycol) (PEG) and biotin-PEG to primary amine groups of proteins using N-hydroxysuccinimide reactions. Four types of protein conjugates were successfully prepared: PEGylated LZ (PEG-LZ), PEGylated BSA (PEG-BSA), biotin-PEG-conjugated LZ (Bio-PEG-LZ), and biotin-PEG-conjugated BSA (Bio-PEG-BSA). PEG-LZ and Bio-PEG-LZ exhibited a lower intracellular uptake than that of LZ in A549 human lung cancer cells (in a two-dimensional culture). However, Bio-PEG-BSA showed significantly improved intracellular delivery as compared to that of PEG-BSA and BSA, probably because of favorable interactions with cells via biotin receptors. For A549/fibroblast coculture spheroids, PEG-LZ and PEG-BSA exhibited significantly decreased tissue penetration as compared with that of unmodified proteins. However, Bio-PEG-BSA showed tissue penetration comparable to that of unmodified BSA. In addition, citraconlyated LZ (Cit-LZ) showed reduced spheroid penetration as compared to that of LZ, probably owing to a decrease in protein charge. Taken together, chemical conjugation of targeting ligands-PEG to anionic proteins could be a promising strategy to improve intracellular delivery and in vivo activity, whereas modifications of cationic proteins should be more delicately designed.

Keywords:  PEGylation; anionic protein; biotinylation; cationic protein; intracellular uptake; spheroid penetration

DOI:  https://doi.org/10.4014/jmb.2207.07058
J Nanobiotechnology. 2022 Aug 31. 20(1): 391

Synthesis of doxorubicin-loaded peptosomes hybridized with gold nanorod for targeted drug delivery and CT imaging of metastatic breast cancer.

Maliheh Hasannia, Khalil Abnous, Seyed Mohammad Taghdisi, Sirous Nekooei, Mohammad Ramezani, Mona Alibolandi.

   BACKGROUND: Cancer nanomedicines based on synthetic polypeptides have attracted much attention due to their superior biocompatibility and biodegradability, stimuli responsive capability through secondary conformation change, adjustable functionalities for various cargos such as peptides, proteins, nucleic acids and small therapeutic molecules. Recently, a few nanoformulations based on polypeptides comprising NK105, NC6004, NK911, CT2103, have entered phase I-III clinical trials for advanced solid tumors therapy. In the current study, we prepared polypeptide-based vesicles called peptosome via self-assembly of amphiphilic polypeptide-based PEG-PBLG diblock copolymer.
RESULTS: In this regard, poly(γ-benzyl L-glutamate (PBLG) was synthesized via ring opening polymerization (ROP) of γ-benzyl L-glutamate-N-carboxyanhydride (BLG-NCA) using N-hexylamine as initiator. Then amine-terminated PBLG was covalently conjugated to heterofuctional maleimide PEG-carboxylic acid or methyl-PEG-carboxylic acid. The PEG-PBLG peptosomes were prepared through double emulsion method for the co-delivery of doxorubicin.HCl and gold nanorods as hydrophilic and hydrophobic agents in interior compartment and membrane of peptosomes, respectively (Pep@MUA.GNR-DOX) that DOX encapsulation efficiency and loading capacity were determined 42 ± 3.6 and 1.68 ± 3.6. Then, theranostic peptosomes were decorated with thiol-functionalized EpCAM aptamer throught thiol-maleimide reaction producing Apt-Pep@MUA.GNR-DOX for targeted delivery. The non-targeted and targeted peptosomes showed 165.5 ± 1.1 and 185 ± 4.7 nm diameters, respectively while providing sustained, controlled release of DOX. Furthermore, non-targeted and targeted peptosomes showed considerable serum stability. In vitro study on MCF-7 and 4T1 cells showed significantly higher cytotoxicity for Apt-Pep@MUA.GNR-DOX in comparison with Pep@MUA.GNR-DOX while both system did not show any difference in cytotoxicity against CHO cell line. Furthermore, Apt-Pep@MUA.GNR-DOX illustrated higher cellular uptake toward EpCAM-overexpressing 4T1 cells compared to Pep@MUA.GNR-DOX. In preclinical stage, therapeutic and diagnostic capability of the prepared Pep@MUA.GNR-DOX and Apt-Pep@MUA.GNR-DOX were investigated implementing subcutaneous 4T1 tumor model in BALB/c mice. The obtained data indicated highest therapeutic index for Apt-Pep@MUA.GNR-DOX compared to Pep@MUA.GNR-DOX and free DOX. Moreover, the prepared system showed capability of CT imaging of tumor tissue in 4T1 tumorized mice through tumor accumulation even 24 h post-administration.
CONCLUSION: In this regard, the synthesized theranostic peptosomes offer innovative hybrid multipurpose platform for fighting against breast cancer.

Keywords:  Breast cancer; Doxorubicin; Gold nanorod; Peptosome; Theranostics

DOI:  https://doi.org/10.1186/s12951-022-01607-2
Front Aging Neurosci. 2022 ;14 960246

QbD-based rivastigmine tartrate-loaded solid lipid nanoparticles for enhanced intranasal delivery to the brain for Alzheimer's therapeutics.

Deepshi Arora, Shailendra Bhatt, Manish Kumar, Ravinder Verma, Yugam Taneja, Nikita Kaushal, Abhishek Tiwari, Varsha Tiwari, Athanasios Alexiou, Sarah Albogami, Saqer S Alotaibi, Vineet Mittal, Rajeev K Singla, Deepak Kaushik, Gaber El-Saber Batiha.

  Alzheimer's disease (AD) is a neurodegenerative disease that affects a wide range of populations and is the primary cause of death in various countries. The treatment of AD is still restricted to oral conventional medicines that act only superficially. Fabrication of intranasal solid lipid nanoparticulate system for the uptake of therapeutic agents will act as a convincing approach with limited off-site toxicity and increased pharmacological activity. The objective of this study was to formulate, optimize, and evaluate the efficiency of rivastigmine tartrate (RT)-loaded intranasal solid lipid nanoparticles (SLNs) employing the solvent-evaporation diffusion method. To optimize the formulation parameters, the central composite design (CCD) was used. Lipid concentration (X1) and surfactant concentration (X2) were considered to be independent variables, while particle size (Y1), percentage entrapment efficiency (Y2), and percentage drug release (Y3) were considered as responses. The solid lipid was glyceryl monostearate, while the surfactant was polysorbate 80. The optimized formulation has a particle size of 110.2 nm, % entrapment efficiency of 82.56%, and % drug release of 94.86%. The incompatibility of drug excipients was established by differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR). Nasal histopathology tests on sheep mucosa revealed that the developed SLNs were safe to utilize for intranasal delivery with no toxicity. Ex vivo permeation investigations revealed that the flux and diffusion coefficients for RT solid lipid nanoparticles and RT solution were 3.378 g/cm2 /h and 0.310-3 cm2 /h, respectively. Stability studies demonstrated that the developed SLNs were stable when stored under various storage conditions. The viability and vitality of adopting a lipid particle delivery system for improved bioavailability via the intranasal route were also established in the in vivo pharmacokinetic investigations. According to the histopathological and pharmacokinetic investigations, the developed formulations were safe, non-lethal, efficient, and robust. These results suggest the potentiality provided by rivastigmine tartrate-loaded solid lipid nanoparticles for nasal delivery.

Keywords:  Alzheimer; CCD; ex vivo; intranasal; optimization; pharmacokinetics; rivastigmine

DOI:  https://doi.org/10.3389/fnagi.2022.960246