Functionalized bioengineered spider silk spheres improve nuclease resistance and activity of oligonucleotide therapeutics providing a strategy for cancer treatment

Abstract

Cell-selective delivery and sensitivity to serum nucleases remain major hurdles to the clinical application of RNA-based oligonucleotide therapeutics, such as siRNA. Spider silk shows great potential as a biomaterial due to its biocompatibility and biodegradability. Self-assembling properties of silk proteins allow for processing into several different morphologies such as fibers, scaffolds, films, hydrogels, capsules and spheres. Moreover, bioengineering of spider silk protein sequences can functionalize silk by adding peptide moieties with specific features including binding or cell recognition domains. We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel oligonucleotide delivery system that can be utilized to improve pharmacokinetics of RNA-based therapeutics, such as CpG-siRNA. The MS2 bioengineered silk was functionalized with poly-lysine domain (KN) to generate hybrid silk MS2KN. CpG-siRNA efficiently bound to MS2KN in contrary to control MS2. Both MS2KN complexes and spheres protected CpG-siRNA from degradation by serum nucleases. CpG-siRNA molecules encapsulated into MS2KN spheres were efficiently internalized and processed by TLR9-positive macrophages. Importantly, CpG-STAT3siRNA loaded in silk spheres showed delayed and extended target gene silencing compared to naked oligonucleotides. The prolonged Stat3 silencing resulted in the more pronounced downregulation of interleukin 6 (IL-6), a proinflammatory cytokine and upstream activator of STAT3, which limits the efficacy of TLR9 immunostimulation. Our results demonstrate the feasibility of using spider silk spheres as a carrier of therapeutic nucleic acids. Moreover, the modified kinetic and activity of the CpG-STAT3siRNA embedded into silk spheres is likely to improve immunotherapeutic effects in vivo.

Drug affinity and targeted delivery: double functionalization of silk spheres for controlled doxorubicin delivery into Her2-positive cancer cells

“…2c). The DOX peptide did not affect the self-assembly properties of MS2 silk and the process of sphere formation; similar results were reported previously for different functional peptides [21,26].…”

Silk Materials Functionalized via Genetic Engineering for Biomedical Applications

“…Unmodified silk-based drug delivery systems address only some of these aspects. Although there are known examples of silk carriers that are non-toxic and biodegradable and can effectively protect their cargo from degradation [ 54 , 55 ], these unmodified silk carriers did not allow for targeted delivery and showed negligible cellular uptake [ 54 , 56 ]. Proposed anti-cancer therapies utilizing unmodified silk fibroin carriers for the delivery of small drugs such as doxorubicin (Dox) [ 57 ] paclitaxel (PTX) [ 58 ], curcumin [ 59 ] or cisplatin [ 60 ] assumed that the deposition of carriers would occur at the tumor site due to the enhanced permeability and retention (EPR) effect.…”

“…The internalization of silk-based carriers into cells can be improved by the introduction of the proper functional peptide into the bioengineered silk. The addition of poly-lysine (KKK KKKKKKKKKKKK) or poly-arginine blocks (RRRRRRRR) significantly increased the uptake of silk spheres into cells [ 51 , 55 , 62 ]. The positive charge of these residues also allowed for the binding of nucleic acids through interactions with the negatively charged phosphate backbone of nucleic acids [ 55 , 62 ].…”

“…The addition of poly-lysine (KKK KKKKKKKKKKKK) or poly-arginine blocks (RRRRRRRR) significantly increased the uptake of silk spheres into cells [ 51 , 55 , 62 ]. The positive charge of these residues also allowed for the binding of nucleic acids through interactions with the negatively charged phosphate backbone of nucleic acids [ 55 , 62 ]. The presence of such peptides did not impair the ability of MS2 (based on MaSp2 spidroin from N. clavipes ) and 6-mer (based on MaSp1 spidroin from N. clavipes ) silk to self-assemble, and these functionalized silks were able to form spheres or polyplexes with nucleic acids [ 55 , 62 ].…”

<p>Cellular uptake, intracellular distribution and degradation of Her2-targeting silk nanospheres</p>

“…The potential application of particles made of silk of various origin as drug carriers has been evaluated in vitro. 12,13,17,[19][20][21][22][23] Several studies to characterize the silk particles degradation have been conducted. Lysosomal inhibitors were used to study their effect on drug release from silkworm silk spheres and thus as an indirect indication of sphere degradation.…”