A coacervate-based platform for growth factor delivery

Growth factors participating in a variety of biological processes have great potential in regenerative medicine. However, unprotected growth factors degrade quickly and have little efficacy at tissue repair. Delivery of growth factors with different vehicles has been examined to prolong the half-lives of growth factors and therefore increase its therapeutic efficacy. After decades of research, controlled delivery of growth factor still faces some significant limitations, and none has reached clinical translation. Heparin, a highly sulfated macromolecule, is used as an anticoagulant clinically. In addition, it has high affinity to a large number of biomolecules, including many growth factors. The interaction between heparin and heparin-binding growth factors is known to adjust their conformation, protect them from proteolytic degradation and regulate their activities. Incorporation of heparin in growth factor delivery is consequently a strategy to potentiate the bioactivity of growth factors. Currently, most approaches used to immobilize heparin on the delivery vehicles rely on covalent modification of heparin that may alter its inherent properties. To maximize the efficacy of heparin, we developed a coacervate-based delivery platform in which heparin is utilized to complex with a polycation without any modification. The polycation neutralizes the negative charges of heparin and precipitates it out of solution. This approach allows spatiotemporal control of the release of heparin-binding growth factors. This dissertation covers the design, production, characterization and application of heparin-based coacervate in controlled release of growth factors

Extension of the crRNA enhances Cpf1 gene editing in vitro and in vivo.

Engineering of the Cpf1 crRNA has the potential to enhance its gene editing efficiency and non-viral delivery to cells. Here, we demonstrate that extending the length of its crRNA at the 5 end can enhance the gene editing efficiency of Cpf1 both in cells and in vivo. Extending the 5 end of the crRNA enhances the gene editing efficiency of the Cpf1 RNP to induce non-homologous end-joining and homology-directed repair using electroporation in cells. Additionally, chemical modifications on the extended 5 end of the crRNA result in enhanced serum stability. Also, extending the 5 end of the crRNA by 59 nucleotides increases the delivery efficiency of Cpf1 RNP in cells and in vivo cationic delivery vehicles including polymer nanoparticle. Thus, 5 extension and chemical modification of the Cpf1 crRNA is an effective method for enhancing the gene editing efficiency of Cpf1 and its delivery in vivo

Aptamer photoregulation in vivo

The in vivo application of aptamers as therapeutics could be improved by enhancing target-specific accumulation while minimizing off-target uptake. We designed a light-triggered system that permits spatiotemporal regulation of aptamer activity in vitro and in vivo. Cell binding by the aptamer was prevented by hybridizing the aptamer to a photo-labile complementary oligonucleotide. Upon irradiation at the tumor site, the aptamer was liberated, leading to prolonged intratumoral retention. The relative distribution of the aptamer to the liver and kidney was also significantly decreased, compared to that of the free aptamer.National Institutes of Health (U.S.) (Grant GM073626

Control Growth Factor Release Using a Self-Assembled [polycation∶heparin] Complex

The importance of growth factors has been recognized for over five decades; however their utilization in medicine has yet to be fully realized. This is because free growth factors have short half-lives in plasma, making direct injection inefficient. Many growth factors are anchored and protected by sulfated glycosaminoglycans in the body. We set out to explore the use of heparin, a well-characterized sulfated glycosaminoglycan, for the controlled release of fibroblast growth factor-2 (FGF-2). Heparin binds a multitude of growth factors and maintains their bioactivity for an extended period of time. We used a biocompatible polycation to precipitate out the [heparin∶FGF-2] complex from neutral buffer to form a release matrix. We can control the release rate of FGF-2 from the resultant matrix by altering the molecular weight of the polycation. The FGF-2 released from the delivery complex maintained its bioactivity and initiated cellular responses that were at least as potent as fresh bolus FGF-2 and fresh heparin stabilized FGF-2. This new delivery platform is not limited to FGF-2 but applicable to the large family of heparin-binding growth factors

RNA therapeutics – The potential treatment for myocardial infarction

© 2016 The Japanese Society for Regenerative Medicine. RNA therapeutics mainly control gene expression at the transcript level. In contrast to conventional gene therapy which solely increases production of a protein, delivered RNAs can enhance, reduce or abolish synthesis of a particular protein, which control its relevant activities in a more diverse fashion. Thus, they hold promise to treat many human diseases including myocardial infarction (MI). MI is a serious health burden that causes substantial morbidity and mortality. An unmet clinical need for treating MI is the recovery of cardiac function, which requires regeneration of the functional tissues including the vasculature, nerves, and myocardium. Several classes of RNA therapeutics have been investigated in preclinical MI models, and the results have demonstrated their benefits and encourage their future development. In this review, we summarize the common RNA therapeutic approaches and highlight their application in MI therapy

The bioactivity of the FGF-2 released from the [PAGS∶heparin] delviery matrix is higher than bolus FGF-2 and matches that of heparin-protected FGF-2.

<p>Note that the growth factor in the release media were 1 and 3 days old, whereas the positive controls were fresh. [A] The mitogenic potency of the released FGF-2 from days 1 and 3 examined using HUVECs indicated that the bioactivity of FGF-2 is well maintained and matched that of the fresh heparin-protected FGF-2. [B] The released FGF-2 demonstrated ability to stimulate endothelial tube formation as well as fresh heparin-protected FGF-2 and is the only group that was statistically significantly better than blous FGF-2 in HAECs. Representative phase contrast micrographs of HUVECs incubated in: [C] no FGF-2, [D] fresh bolus FGF-2, [E] fresh [heparin∶FGF-2], and [F] [PAGS∶heparin∶FGF-2] release media. Statistical significance between the negative control and other experimental groups was noted as “*”, p<0.05. <b>#</b> denotes the statistical significance between an experimental group and the bolus FGF-2 group, p<0.05.</p

Release kinetics of FGF-2 in PBS and in the presence of serum proteins.

<p>[A] Release kinetics as examined by measuring the amount of ¹²⁵I-FGF-2 released from complexes. The percent of FGF-2 released from complexes was monitored over 28 days. Two different molecular weight species of PAGS were used to characterize whether release kinetics were controllable. The LMW species of PAGS released nearly 20% of its loaded growth factor, while the HMW species of PAGS released approximately 50% of incorporated growth factor over the same period of time. [B] The addition of BSA had minor impact on the release FGF-2, which is consistent with the low affinity of BSA with heparin. On the other hand, negatively charged serum proteins in FBS formed precipitate surrounding the delivery matrix, which decreased the FGF-2 release rate.</p

Our delivery strategy was inspired by the interaction among growth factor, heparin and growth factor receptor.

<p><i>Left</i>, the crystal structure of the [FGF∶Heparin∶FGFR] complex kindly provided by Dr. Pellegrini. The proteins are shown as coils and heparin as a stick model. The heparin-binding domains of FGFR and FGF are highlighted in pink and yellow respectively. Both analyses showed that the heparin-binding regions contain a high density of positively charged amino acid residues such as arginine. <i>Right</i>, a possible model of the matrix formed by ionic interactions between an arginine-based synthetic polycation and a [heparin∶growth factor] complex.</p

Complex loading efficiency and capacity was investigated using ¹²⁵I-FGF-2.

<p>[A] Loading efficiencies of different molecular weight PAGS was investigated for different [PAGS∶heparin] ratios. The higher molecular weight PAGS was more efficient at incorporating FGF-2 at all [PAGS∶heparin] ratios than the lower molecular weight polymer. A ratio of [35∶1] was the most efficient at incorporating FGF-2 for both molecular weight species. [B] The loading capacity of complexes was investigated for a [35∶1] ratio of low molecular weight PAGS. This ratio demonstrated a loading efficiency of 50% for all amounts of FGF-2. Statistical significance between [35∶1] and other ratios was noted as “*”, p<0.05.</p

The interaction between PAGS and heparin.

<p>[A] The binding of PAGS and heparin resulted in a white precipitate when combined in an aqueous solution. [B] The titration of heparin using PAGS as monitored by zeta potential measurements. The complex was nearly neutral at a 35/1 mass ratio of PAGS/heparin. [C] SEM images revealed the [PAGS∶heparin] complexes as a matrix composed of fibers and sheets, and beads (1000×). [D] Higher magnification (25000×) revealed that many of the fiber are sub-micron in diameter and the beads were in fact rings.</p