Genetic and Covalent Protein Modification Strategies to Facilitate Intracellular Delivery

Horn, Justin M.; Obermeyer, Allie C.

doi:10.1021/acs.biomac.1c00745

Cited by 28 publications

(27 citation statements)

References 238 publications

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“…28,29 But due to the shortcomings of the physical methods such as inefficient, not scalable, and cytotoxic, it is hard to translate in vivo intracellular delivery of protein therapeutics. 30 With the development of nanotechnology, a new generation to replace the physical methods of delivery strategies with multiple functions (e.g., tumor targeting, deep tumor penetration, and effective cellular uptake) was come up to address these limitations.…”

Section: Strategies For Intracellular Protein Deliverymentioning

confidence: 99%

Receptor-Targeted Dual pH-Triggered Intracellular Protein Transfer

Lyu

Yazdi

Lin

et al. 2022

ACS Biomater. Sci. Eng.

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Protein therapeutics, including monoclonal antibodies, cytokines, transcription factors, enzymes, and peptides, play an essential role against cancer, infectious diseases, cardiovascular disorders, immunological diseases, and metabolic disorders, resulting in brilliant successes. [9][10][11] Compared with chemotherapy, the critical function of protein therapeutics in treating diseases that lack effective therapeutic options relies on the unique advantages of (i) abundant species; (ii) complex set of biological functions; (iii) high specificity; (iv) fewer adverse effects; (v) excellent biocompatibility and biodegradability; and (vi) inherent amino, carboxyl, and hydroxyl groups for chemical conjugations. 12 Proteins, in contrast to genetic drugs, act on their targets in a way that is both more direct and more specific. This allows them to regulate biological processes in a way that eliminates the risk of permanent gene mutation, off-target effects brought on by persistent gene expression, and the possibility of cancer development. 11 Major challenges of protein therapeuticsThe progress path toward clinical application of proteins is neither straightforward nor uncomplicated, for example, due to the limited administration routes, low bioavailability of proteins in circulation, and host immune responses that may result in the inactivation of proteins before reaching their sites of action. 13 Therefore, biotechnologists have employed tremendous efforts to overcome the delivery drawbacks 2, 14, 15 to protect them from denaturation and degradation, facilitate tumor-targeted delivery, and control the protein release/activity in targeted sites. 16,17 Most protein medicines now available on the market were created based on extracellular targets. These extracellular targets include cell membrane proteins (PD1, HER2, CD20, etc.) and secretory proteins (TNFα, IL12, VEGF, etc.). 11,18 Despite the success of current protein products that mostly address extracellular targets, efficient cytosolic delivery of Ju, E. et al. 36 first reported that gold nanoclusters (AuNCs) can self-assemble with Streptococcus pyogenes Cas9 (SpCas9) protein under physiological conditions, and the complexes (SpCas9-AuNCs) efficiently deliver SpCas9 protein into the cell nucleus.SpCas9-AuNCs could be disassembled at a lower pH 4.5 but are stable at a higher pH 7.4. SpCas9 is delivered into cells and the cell nucleus, where it performs its cleavage function, through the assembly disassembly process. Furthermore, the E6 oncogene was effectively knockout by self-assembled SpCas9-AuNCs nanoparticles after the HPV18 E6 sgRNA transfected into cervical cancer cells. Consequently, this causes the tumorsuppressing protein p53 to resume its normal activity and induces apoptosis in cervical cancer cells but had little effect on the normal cells. Because of their unique characteristics, SpCas9-AuNCs are an intriguing biomaterial for the treatment of cancer.Nanogels are water-rich nanoscale three-dimensional polymer networks. Because of this, it is simple for nan...

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Section: Strategies For Intracellular Protein Deliverymentioning

confidence: 99%

Receptor-Targeted Dual pH-Triggered Intracellular Protein Transfer

Lyu

Yazdi

Lin

et al. 2022

ACS Biomater. Sci. Eng.

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“…Hundreds of protein therapeutics have been approved by the FDA for clinical use, and five of the top ten best-selling drugs were proteins last year. 2,3 Despite the progress, all of the marketed protein drugs function extracellularly due to their limited membrane permeability. 4 Developing efficient intracellular protein transfer carriers plays an important role in the development of cytosolic protein-related biotherapeutics and biotechnology.…”

Section: Introductionmentioning

confidence: 99%

Histidine-based coordinative polymers for efficient intracellular protein delivery via enhanced protein binding, cellular uptake, and endosomal escape

et al. 2023

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“…3,4 Two major strategies have been studied to improve intracellular protein delivery: increasing the hydrophobicity of the protein and increasing the cationic charge on the protein. 5 It has previously been demonstrated that hydrophobic proteins can translocate across the cell membrane, most likely due to favorable hydrophobic interactions with the lipid bilayer. Mix et al were able to deliver an esterified green fluorescent protein (GFP) directly to the cytosol.…”

Section: Introductionmentioning

confidence: 99%

Protein charge parameters that influence stability and cellular internalization of polyelectrolyte complex micelles

Kapelner

Obermeyer

2022

Preprint

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Proteins are an important class of biologics, but there are several recurring challenges to address when designing protein-based therapeutics. These challenges include: the propensity of proteins to aggregate during formulation, relatively low loading in traditional hydrophobic delivery vehicles, and inefficient cellular uptake. This last criterion is particularly challenging for anionic proteins as they cannot cross the anionic plasma membrane. Here we investigated the complex coacervation of anionic proteins with a block copolymer of opposite charge to form polyelectrolyte complex (PEC) micelles for use as a protein delivery vehicle. Using genetically modified variants of the model protein green fluorescent protein (GFP), we evaluated the role of protein charge and charge localization in the formation and stability of PEC micelles. A neutral-cationic block copolymer, POEGMA79-b-qP4VP175, was prepared via RAFT polymerization for complexation and microphase separation with the panel of engineered anionic GFPs. We found that isotropically supercharged proteins formed micelles at higher ionic strength relative to protein variants with charge localized to a polypeptide tag. We then studied GFP delivery by PEC micelles and found that they effectively delivered the protein cargo to mammalian cells. However, cellular delivery varied as a function of protein charge and charge distribution and we found an inverse relationship between the PEC micelle critical salt concentration and delivery efficiency. This model system has highlighted the potential of polyelectrolyte-complexes to deliver anionic proteins intracellularly as well as the importance of correlating solution structure and desired functional activity.

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Genetic and Covalent Protein Modification Strategies to Facilitate Intracellular Delivery

Cited by 28 publications

References 238 publications

Receptor-Targeted Dual pH-Triggered Intracellular Protein Transfer

Receptor-Targeted Dual pH-Triggered Intracellular Protein Transfer

Histidine-based coordinative polymers for efficient intracellular protein delivery via enhanced protein binding, cellular uptake, and endosomal escape

Protein charge parameters that influence stability and cellular internalization of polyelectrolyte complex micelles

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