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Nearly
20% of HER2-positive breast cancers develop resistance to
HER2-targeted therapies requiring the use of advanced therapies. Silencing
RNA therapy may be a powerful modality for treating resistant HER2
cancers due to its high specificity and low toxicity. However, the
systemic administration of siRNAs requires a safe and efficient delivery
platform because of siRNA’s low stability in physiological
fluids, inefficient cellular uptake, immunoreactivity, and rapid clearance.
We have developed theranostic polymeric vesicles to overcome these
hurdles for encapsulation and delivery of small functional molecules
and PARP1 siRNA for in vivo delivery to breast cancer tumors. The
100 nm polymer vesicles were assembled from biodegradable and non-ionic
poly(N-vinylpyrrolidone)14-block-poly(dimethylsiloxane)47-block-poly(N-vinylpyrrolidone)14 triblock copolymer PVPON14−PDMS47−PVPON14 using
nanoprecipitation and thin-film hydration. We demonstrated that the
vesicles assembled from the copolymer covalently tagged with the Cy5.5
fluorescent dye for in vivo imaging could also encapsulate the model
drug with high loading efficiency (40%). The dye-loaded vesicles were
accumulated in tumors after 18 h circulation in 4TR breast tumor-bearing
mice via passive targeting. We found that PARP1 siRNA encapsulated
into the vesicles was released intact (13%) into solution by the therapeutic
ultrasound treatment as quantified by gel electrophoresis. The PARP1
siRNA-loaded polymersomes inhibited the proliferation of MDA-MB-361TR
cells by 34% after 6 days of treatment by suppressing the NF-kB signaling
pathway, unlike their scrambled siRNA-loaded counterparts. Finally,
the treatment by PARP1 siRNA-loaded vesicles prolonged the survival
of the mice bearing 4T1 breast cancer xenografts, with the 4-fold
survival increase, unlike the untreated mice after 3 weeks following
the treatment. These biodegradable, non-ionic PVPON14−PDMS47−PVPON14 polymeric nanovesicles capable
of the efficient encapsulation and delivery of PARP1 siRNA to successfully
knock down PARP1 in vivo can provide an advanced platform for the
development of precision-targeted therapeutic carriers, which could
help develop highly effective drug delivery nanovehicles for breast
cancer gene therapy.
Phase separation plays crucial roles in both sustaining cellular function and perpetuating disease states. Despite extensive studies, our understanding of this process is hindered by low solubility of phase-separating proteins. One example of this is found in SR and SR-related proteins. These proteins are characterized by domains rich in arginine and serine (RS domains), which are essential to alternative splicing and in vivo phase separation. However, they are also responsible for a low solubility that has made these proteins difficult to study for decades. Here, we solubilize the founding member of the SR family, SRSF1, by introducing a peptide mimicking RS repeats as a co-solute. We find that this RS-mimic peptide forms interactions similar to those of the protein's RS domain. Both interact with a combination of surface-exposed aromatic residues and acidic residues on SRSF1's RNA Recognition Motifs (RRMs) through electrostatic and cation-pi interactions. Analysis of RRM domains from human SR proteins indicates that these sites are conserved across the protein family. In addition to opening an avenue to previously unavailable proteins, our work provides insight into how SR proteins phase separate and participate in nuclear speckles.
Phase separation plays crucial roles in both sustaining cellular function and perpetuating disease states. Despite extensive studies, our understanding of this process is hindered by low solubility of phase-separating proteins. One example of this is found in SR proteins. These proteins are characterized by domains righ in arginine and serine (RS domains), which are essential to alternative splicing, in vivo phase separation, and a low solubility that has made these proteins difficult to study for decades. Here, we solubilize the founding member of the SR family, SRSF1, by introducing a peptide mimicking RS repeats as a cosolute. We find that this RS-mimic peptide forms interactions similar to those of the protein's RS domain. Both interact with a combination of surface-exposed aromatic residues and acidic residues on SRSF1's RNA Recognition Motifs (RRMs) through simultaneous electrostatic and cation-pi bonding. Analysis of RRM domains spanning the human proteome indicates that RRM domains involved in phase separation have more exposed aromatic residues. Further, in phase-separating proteins containing RS repeats, exposed aromatic residues are frequently surrounded by acidic residues. Our work provides insight into how SR proteins phase separate and opens an avenue to a range of previously unavailable proteins.
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