Victoria Kett

Development and characterization of self-assembling nanoparticles using a bio-inspired amphipathic peptide for gene delivery

The design of a non-viral gene delivery vehicle capable of delivering and releasing a functional nucleic acid cargo intracellularly remains a formidable challenge. For systemic gene therapy to be successful a delivery vehicle is required that protects the nucleic acid cargo from enzymatic degradation, extravasates from the vasculature, traverses the cell membrane, disrupts the endosomal vesicles and unloads the cargo at its destination site, namely the nucleus for the purposes of gene delivery. This manuscript reports the extensive investigation of a novel amphipathic peptide composed of repeating RALA units capable of overcoming the biological barriers to gene delivery both in vitro and in vivo. Our data demonstrates the spontaneous self-assembly of cationic DNA-loaded nanoparticles when the peptide is complexed with pDNA. Nanoparticles were <100nm, were stable in the presence of serum and were fusogenic in nature, with increased peptide α-helicity at a lower pH. Nanoparticles proved to be non-cytotoxic, readily traversed the plasma membrane of both cancer and fibroblast cell lines and elicited reporter-gene expression following intravenous delivery in vivo. The results of this study indicate that RALA presents an exciting delivery platform for the systemic delivery of nucleic acid therapeutics.

Hydrogel-Forming Microneedles Prepared from ‘‘Super Swelling’’ Polymers Combined with Lyophilised Wafers for Transdermal Drug Delivery

We describe, for the first time, hydrogel-forming microneedle arrays prepared from “super swelling” polymeric compositions. We produced a microneedle formulation with enhanced swelling capabilities from aqueous blends containing 20% w/w Gantrez S-97, 7.5% w/w PEG 10,000 and 3% w/w Na2CO3 and utilised a drug reservoir of a lyophilised wafer-like design. These microneedle-lyophilised wafer compositions were robust and effectively penetrated skin, swelling extensively, but being removed intact. In in vitro delivery experiments across excised neonatal porcine skin, approximately 44 mg of the model high dose small molecule drug ibuprofen sodium was delivered in 24 h, equating to 37% of the loading in the lyophilised reservoir. The super swelling microneedles delivered approximately 1.24 mg of the model protein ovalbumin over 24 h, equivalent to a delivery efficiency of approximately 49%. The integrated microneedle-lyophilised wafer delivery system produced a progressive increase in plasma concentrations of ibuprofen sodium in rats over 6 h, with a maximal concentration of approximately 179 µg/ml achieved in this time. The plasma concentration had fallen to 71±6.7 µg/ml by 24 h. Ovalbumin levels peaked in rat plasma after only 1 hour at 42.36±17.01 ng/ml. Ovalbumin plasma levels then remained almost constant up to 6 h, dropping somewhat at 24 h, when 23.61±4.84 ng/ml was detected. This work represents a significant advancement on conventional microneedle systems, which are presently only suitable for bolus delivery of very potent drugs and vaccines. Once fully developed, such technology may greatly expand the range of drugs that can be delivered transdermally, to the benefit of patients and industry. Accordingly, we are currently progressing towards clinical evaluations with a range of candidate molecules.

Freeze-dried, mucoadhesive system for vaginal delivery of the HIV microbicide, dapivirine: Optimisation by an artificial neural network.

Dapivirine mucoadhesive gels and freeze-dried tablets were prepared using a 3x3x2 factorial design. An artificial neural network (ANN) with multi-layer perception was used to investigate the effect of hydroxypropyl-methylcellulose (HPMC): polyvinylpyrrolidone (PVP) ratio (X1), mucoadhesive concentration (X2) and delivery system (gel or freeze-dried mucoadhesive tablet, X3) on response variables; cumulative release of dapivirine at 24h (Q(24)), mucoadhesive force (F(max)) and zero-rate viscosity. Optimisation was performed by minimising the error between the experimental and predicted values of responses by ANN. The method was validated using check point analysis by preparing six formulations of gels and their corresponding freeze-dried tablets randomly selected from within the design space of contour plots. Experimental and predicted values of response variables were not significantly different (p>0.05, two-sided paired t-test). For gels, Q(24) values were higher than their corresponding freeze-dried tablets. F(max) values for freeze-dried tablets were significantly different (2-4 times greater, p>0.05, two-sided paired t-test) compared to equivalent gels. Freeze-dried tablets having lower values for X1 and higher values for X2 components offered the best compromise between effective dapivirine release, mucoadhesion and viscosity such that increased vaginal residence time was likely to be achieved.

The effect of polymer coatings on physicochemical properties of spray-dried liposomes for nasal delivery of BSA

This work describes the development of spray dried polymer coated liposomes composed of soy phosphatidylcholine (SPC) and phospholipid dimyristoyl phosphatidylglycerol (DMPG) coated with alginate, chitosan or trimethyl chitosan (TMC), that are able to penetrate through the nasal mucosa and offer enhanced penetration over uncoated liposomes when delivered as a dry powder. All the liposome formulations, loaded with BSA as model antigen, were spray-dried to obtain powder size and liposome size in a suitable range for nasal delivery. Although coating resulted in some reduction in encapsulation efficiency, levels were still maintained between 60% and 69% and the structural integrity of the entrapped protein and its release characteristics were maintained. Coating with TMC gave the best product characteristics in terms of entrapment efficiency, glass transition (T(g)) and mucoadhesive strength, while penetration of nasal mucosal tissue was very encouraging when these liposomes were administered as dispersions although improved results were observed for the dry powders.

Development of polymeric–cationic peptide composite nanoparticles, a nanoparticle-in-nanoparticle system for controlled gene delivery

We report the formulation of novel composite nanoparticles that combine the high transfection efficiency of cationic peptide-DNA nanoparticles with the biocompatibility and prolonged delivery of polylactic acid–polyethylene glycol (PLA-PEG). The cationic cell-penetrating peptide RALA was used to condense DNA into nanoparticles that were encapsulated within a range of PLA-PEG copolymers. The composite nanoparticles produced exhibited excellent physicochemical properties including size <200 nm and encapsulation efficiency >80%. Images of the composite nanoparticles obtained with a new transmission electron microscopy staining method revealed the peptide-DNA nanoparticles within the PLA-PEG matrix. Varying the copolymers modulated the DNA release rate >6 weeks in vitro. The best formulation was selected and was able to transfect cells while maintaining viability. The effect of transferrin-appended composite nanoparticles was also studied. Thus, we have demonstrated the manufacture of composite nanoparticles for the controlled delivery of DNA.

Intravaginal immunization using a novel antigen delivery device elicits robust vaccine antigen-specific systemic and mucosal humoral immune responses

Background While it is relatively easy to elicit antigen-specific serum antibody it is much more difficult to establish meaningful levels of specific antibody at mucosal surfaces, the major route of viral invasion. We sought to determine if mucosal vaccination using topical vaginal application could initiate local antigen-specific immunity, enhance previously existing systemic immunity or re-target responses to the mucosae.

The RALA delivery platform: Altering the biodistribution of nucleic acids in vivo

Statement of the Problem: Nowadays liver cancer has become the second leading cause of cancer related death globally. Systemic therapy with chemotherapeutic agents has severe toxicity to normal cells and the application is limited in pre-clinical study. Researchers have reported that liposomal drug delivery system working as a carrier by encapsulating chemotherapeutic agents into its hydrophobic or hydrophilic parts can enhance the bioavailability and solubility of drugs, facilitate the tumor-specific targeting treatment purpose with surface modification, and further reduce the drug toxicity to normal tissues. However, these delivery systems have not been adopted in clinics. The purpose of this study is to develop cancer-cell specific penetrating peptide modified liposomal delivery system with CTD encapsulated for targeted hepatocellular carcinoma treatment.L delivery represents a fascinating option to limit ubiquitous distribution of systemically, and often chronically, administered drugs used to treat severe pulmonary diseases. Nonetheless, clinical outcomes of inhaled therapies strongly depend on drug ability to deposit along the airways and to overcome barriers imposed by the lungs. In this context, the general aim of our studies is the development of inhalable nanomedicines able to deliver the intact drug in the lungs and to shield its interactions with lung lining fluids while enhancing drug availability at the cell target. This objective has been pursued through the design and production of differently engineered nanoparticulate systems with increasing levels of complexity, driven by technological and biological design rules. Some examples, such as drug nanocrystals, micelles and biodegradable poly(lactide-co-glycolide) (PLGA) nanoparticles, will be discussed highlighting how the most appropriate formulation approach can be selected only taking into account the distinct physico-chemical profile of the drug under investigation (e.g., molecular weight, solubility, stability) and the peculiarities of the lung pathology (e.g., cystic fibrosis, lung cancer). Surface engineering of nanocarriers with either polymers or phospholipids turns out as crucial to face the current challenge of overcoming lung barriers, especially mucus. Last but not least, in vitro/in vivo studies represent a critical step to select the best formulation to candidate for further development.