skip to main content

Explore Research Products in the NSF-PAR

Title: Connectosomes for Direct Delivery of siRNA into the Cytoplasm.
Introduction: The plasma membrane protects a cell from the extracellular environment. As such it presents an obstacle that therapeutics needs to traverse in order to achieve efficacy. For example, small interfering RNAs (siRNAs) need to be delivered to the cytoplasm, where they can interact with the RNA interference machinery and initiate gene silencing. However, these macromolecules have poor membrane permeability, largely limiting their therapeutic potential. To address this challenge, current strategies involve encapsulating siRNAs into nanoparticles. However, upon cellular uptake, these nanoparticles are trapped in endosomes, which lack access to the cytoplasm. Towards developing an alternative strategy that provides direct access to the cytoplasm, we have been inspired by the unique capabilities of gap junctions to establish passageways between the cytoplasm of neighboring cells. Specifically, six connexins hexamerize to form a connexon hemichannel. Two hemichannels from neighboring cells dock to each other to form a complete gap junction channel, facilitating the exchange of molecular cargoes such as ions and siRNA. Therefore, incorporating the gap junction network into therapeutic delivery materials has the potential to enhance the delivery efficiency of siRNAs by directly depositing siRNAs into the cytoplasm.  more » « less
Award ID(s):
1757885
NSF-PAR ID:
10138554
Author(s) / Creator(s):
; ; ;
Date Published:
Journal Name:
2019 BMES Conference Proceedings - REU Abstract Accepted Poster
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ( , Advanced Materials)
    Abstract

    Despite the promise of ribonucleic acid interference therapeutics, the delivery of oligonucleotides selectively to diseased tissues in the body, and specifically to the cellular location in the tissues needed to provide optimal therapeutic outcome, remains a significant challenge. Here, key material properties and biological mechanisms for delivery of short interfering RNAs (siRNAs) to effectively silence target‐specific cells in vivo are identified. Using porous silicon nanoparticles as the siRNA host, tumor‐targeting peptides for selective tissue homing, and fusogenic lipid coatings to induce fusion with the plasma membrane, it is shown that the uptake mechanism can be engineered to be independent of common receptor‐mediated endocytosis pathways. Two examples of the potential broad clinical applicability of this concept in a mouse xenograft model of ovarian cancer peritoneal carcinomatosis are provided: silencing theRev3lsubunit of polymerase Pol ζ to impair DNA repair in combination with cisplatin; and reprogramming tumor‐associated macrophages into a proinflammatory state.

     
    more » « less
  2. ; ; ; ; ; ; ; ; ( , Small)
    Abstract

    The therapeutic potential of small interfering RNAs (siRNAs) is limited by their poor stability and low cellular uptake. When formulated as spherical nucleic acids (SNAs), siRNAs are resistant to nuclease degradation and enter cells without transfection agents with enhanced activity compared to their linear counterparts; however, the gene silencing activity of SNAs is limited by endosomal entrapment, a problem that impacts many siRNA‐based nanoparticle constructs. To increase cytosolic delivery, SNAs are formulated using calcium chloride (CaCl2) instead of the conventionally used sodium chloride (NaCl). The divalent calcium (Ca2+) ions remain associated with the multivalent SNA and have a higher affinity for SNAs compared to their linear counterparts. Importantly, confocal microscopy studies show a 22% decrease in the accumulation of CaCl2‐salted SNAs within the late endosomes compared to NaCl‐salted SNAs, indicating increased cytosolic delivery. Consistent with this finding, CaCl2‐salted SNAs comprised of siRNA and antisense DNA all exhibit enhanced gene silencing activity (up to 20‐fold), compared to NaCl‐salted SNAs regardless of sequence or cell line (U87‐MG and SK‐OV‐3) studied. Moreover, CaCl2‐salted SNA‐based forced intercalation probes show improved cytosolic mRNA detection.

     
    more » « less
  3. ; ; ; ; ( , Proceedings of the National Academy of Sciences)
    null (Ed.)
    To realize RNA interference (RNAi) therapeutics, it is necessary to deliver therapeutic RNAs (such as small interfering RNA or siRNA) into cell cytoplasm. A major challenge of RNAi therapeutics is the endosomal entrapment of the delivered siRNA. In this study, we developed a family of delivery vehicles called Janus base nanopieces (NPs). They are rod-shaped nanoparticles formed by bundles of Janus base nanotubes (JBNTs) with RNA cargoes incorporated inside via charge interactions. JBNTs are formed by noncovalent interactions of small molecules consisting of a base component mimicking DNA bases and an amino acid side chain. NPs presented many advantages over conventional delivery materials. NPs efficiently entered cells via macropinocytosis similar to lipid nanoparticles while presenting much better endosomal escape ability than lipid nanoparticles; NPs escaped from endosomes via a “proton sponge” effect similar to cationic polymers while presenting significant lower cytotoxicity compared to polymers and lipids due to their noncovalent structures and DNA-mimicking chemistry. In a proof-of-concept experiment, we have shown that NPs are promising candidates for antiviral delivery applications, which may be used for conditions such as COVID-19 in the future. 
    more » « less
  4. ; ; ; ; ( , ChemMedChem)
    Abstract

    Many intracellular signaling events remain poorly characterized due to a general lack of tools to interfere with “undruggable” targets. Antibodies have the potential to elucidate intracellular mechanisms via targeted disruption of cell signaling cascades because of their ability to bind to a target with high specificity and affinity. However, due to their size and chemical composition, antibodies cannot innately cross the cell membrane, and thus access to the cytosol with these macromolecules has been limited. Herein we describe strategies for accessing the intracellular space with recombinant antibodies mediated by cationic lipid nanoparticles to selectively disrupt intracellular signaling events. Together, our results demonstrate the use of recombinantly produced antibodies, delivered at concentrations of 10 nM, to selectively interfere with signaling driven by a single posttranslational modification. Efficient intracellular delivery of engineered antibodies opens up possibilities for modulation of previously “undruggable” targets, including for potential therapeutic applications.

     
    more » « less
  5. ; ; ; ( , Biotechnology Progress)
    Abstract

    Single‐chain variable fragment (scFv) antibodies have great potential for a range of applications including as diagnostic and therapeutic agents. However, production of scFvs is challenging because proper folding and activity depend on the formation of two intrachain disulfide bonds that do not readily form in the cytoplasm of living cells. Functional expression in bacteria therefore involves targeting to the more oxidizing periplasm, but yields in this compartment can be limiting due to secretion bottlenecks and the relatively small volume compared to the cytoplasm. In the present study, we evaluated an anti‐HER2 scFv, which is specific for human epidermal growth receptor 2 (HER2) overexpressed in breast cancer, for functional expression in the cytoplasm ofEscherichia colistrains BL21(DE3) and SHuffle T7 Express, the latter of which is genetically engineered for cytoplasmic disulfide bond formation. Specifically, we observed much greater solubility and binding activity with SHuffle T7 Express cells, which likely resulted from the more oxidative cytoplasm in this strain background. We also found that SHuffle T7 Express cells were capable of supporting high‐level soluble production of anti‐HER2 scFvs with intact disulfide bonds independent of variable domain orientation, providing further evidence that SHuffle T7 Express is a promising host for laboratory and preparative expression of functional scFv antibodies.

     
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email