The aim of the study was to evaluate a community pharmacy-based cardiovascular risk assessment (VRA) service introduced into two pharmacies in south Wales. A longitudinal methodology was adopted where participants had an initial assessment with a follow-up after 12 months. Body mass index, waist circumference, blood pressure and total/HDL cholesterol levels were measured and the Framingham 10-year cardiovascular risk was estimated and communicated to patients. Demographic details and lifestyle information (smoking, alcohol, diet and exercise) were obtained via self-complete questionnaires at each consultation. A total of 172 individuals accessed the service and had either a brief assessment (n=26) without the calculation of the Framingham score or a full VRA (n=146). Mean age was 60 years (±10.3), 59% were female and 25% (37/146) were at high risk (>20%) of developing cardiovascular disease. High satisfaction with the VRA was obtained via an anonymous questionnaire provided immediately after the initial consultation (74% response rate). The short-term outcomes of the service (including recall of advice, lifestyle improvement and/or making the visit to their GP if they were referred) were reported through a semi-structured telephone interview two weeks after the initial assessment. In total 105/172 (61%) who attended the twelve-month follow-up had results of the two assessments compared using paired Student’s t-test. There was a statistically significant increase in mean HDL 0.08 mmol/L (95% CI 0.02 to 0.14) and a statically significant reduction in mean systolic BP -8.5 mmHg (95% CI -11.0 to -5.9), diastolic BP -7.7 mmHg (95% CI -10.4 to -5.0) and Framingham score -1.07 (95% CI -1.9 to -0.2). A comparison between Framingham and QRISK2 algorithms showed the importance of using the most accurate tool available in estimating cardiovascular risk. This is the first study to investigate short- and longer-term outcomes of a community pharmacy-based VRA service in Wales and provides a basis for future research.

As the global population increases, estimated to reach 9 billion by the year 2050, global food security becomes a priority. A prominent disease implicated in financial loss to the poultry industry, on a global scale, is infectious bursal disease virus (IBDV). Vaccination against IBDV is sub-optimal and difficult to deliver. Therefore it has been highlighted as a key area for the development of an oral vaccine. A highly conserved capsid protein from IBDV (VP2) was identified, and sub-cloned into a bacterial expression cassette. This protein was fused to a potential carrier protein (cholera toxin B chain), previously shown to mediate the exit from the gut lumen into the lamina propria. However, to allow this antigen to reach the mucosal associated lymphoid tissue, the protein antigen must remain in its native conformation through the stomach. This work developed a delivery system to meet this end. By encapsulation within a fatty acid coated, protein adsorbed-solid core drug delivery system (SCDDS), it was shown that a model protein antigen (GST-GFP) could be protected from low pH (i.e. pH 2.0) and proteases. Protease protection was demonstrated against the exposure of myristic acid coated, GST-GFP adsorbed silica, to both protease K (100 μU, 1hour (100% protection)) as well as a simulated in vitro stomach environment (pepsin (0.2 mg) (100% protection)). Having demonstrated protection from proteases at pH 2.0 and pH7.4, it was then shown that GST-GFP could be released from the myristic acid coated silica at pH 8.8 (consistent with the small intestine). As much as ~15% (15 μg) (w/w) GST-GFP was released from the aforementioned system. The evidence supporting this conclusion was drawn from molar ellipticity calculations that showed the proportion of helical structure in relation to regions of beta sheet remained constant, pre-adsorption and post-release (16.9% α-helix, 20.8% β-sheet, 43.3% random coil). Finally, this work has shown that if a recombinant antigen was fused to cholera toxin B chain (but not shiga toxin B chain), it was capable of mediating transcytotic passage across, differentiated, polarised Caco-2 cells (1/1000th input (10 ng)). In conclusion and based upon the evidence provided above, this system warrants further optimisation and investigation to serve as an oral vaccine delivery system to treat IBDV.

The prevalence of Type 2 diabetes mellitus (T2DM) has increased dramatically over the past 10 years in Malaysia due to the modernisation of the country. The most recent national health survey revealed that more than 15.2% of Malaysian adults are suffering from the diseases. Insulin treatment has been demonstrated to play a clinically significant role to improve glycaemic control among selected Type 2 diabetes mellitus (T2DM) patients. However, studies from several local hospitals showed that more than half of T2DM patients are reluctant to initiate insulin treatment. There is an increasing trend for the Malaysian healthcare sector to invest in understanding patients’ health experiences. This qualitative study focused on T2DM patients’ insights about their lived-experience of using insulin treatment as part of their diabetes management. This study aimed to understand the facilitators, which encourage patients to accept insulin treatment and also the psychological, social and behavioural barriers to effective diabetes management. Drawing on interview data with 37 participants, the three main barriers to initiate insulin treatment were worries about inability to handle using insulin, a sense of personal failure and negative perceptions of injections due to past experiences. The facilitators that encourage patients to accept insulin treatment were prior exposure to insulin injections, better side effect profile and wanting a better quality of life. However, there were many obstacles faced by T2DM patients when coping with insulin treatment such as the restriction of lifestyle and concerns about social acceptance. In general, knowledge of T2DM and insulin treatment are still lacking among the Malaysians interviewed in this study. There are still many distorted beliefs and misconceptions about insulin among T2DM patients. At the same time, patients’ concerns and beliefs regarding insulin use are greatly influenced by their experience and support from others. Many participants felt embarrassed and self-conscious when self-injecting insulin in public places. They felt that the Malaysian public often associate the use of injections to drug abuse. Social stigma is one key point, which leads to poor adherence to insulin treatment. Thus it is crucial to increase public awareness about insulin treatment in order to help these patients to be more comfortable about injecting, and also to encourage other people to be more open minded towards insulin treatment. Apart from raising public awareness, there is a need to empower T2DM patients with adequate knowledge through early, simplified, tailored education focusing on the disease nature and the role of insulin. Making them more aware of their health condition and the uses of modern insulin devices at an early stage will better prepare them mentally for insulin therapy.

Adverse drug reactions remain a major health issue with delayed type hypersensitivity reactions developing in a high number of individuals. The cellular immunological processes that underlie drug-specific responses in hypersensitive patients have been previously described; however the involvement of the humoral immune system has not been studied in great detail. Consequently, this thesis explores the nature of the piperacillin-specific B cell response in hypersensitive patients and compares the cellular and humoral immune response that develops in patients with cystic fibrosis (CF) exposed to repeated courses of the drug. Initial studies involved characterization of B cell proliferation, B cell phenotype and the nature of total and drug-specific IgG antibody secretions using peripheral blood mononuclear cells (PBMC) from hypersensitive patients. For comparison PBMCs from 2 groups of individuals were assessed: piperacillin naïve healthy volunteers and piperacillin tolerant patients with CF. ELISA, and ELISpot were used to detect piperacillin-specific B cells responses and IgG secretion. T lymphocyte proliferation was assessed with the lymphocyte transformation test (LTT). T lymphocytes from hypersensitive patients, but not tolerant patients or naïve donors were stimulated to proliferate in the presence of the drug. The peak concentration for T cell activation was 1 mM. Phenotypic assessment of hypersensitive patients B-cells revealed an increase in CD19+CD27+ expression in response to piperacillin treatment in vitro. IgG secreting immortalized B-cell lines also expressed a pure CD19+CD27+ phenotype. Piperacillin stimulation of hypersensitive patient PBMC also led to an increase in the secretion of IgG. In contrast, IgG secretion was not detectable following piperacillin stimulation of PBMC from tolerant patients and healthy controls. Western blotting and mass spectrometric methods were applied to characterize -lactam-protein covalent binding. Bovine serum albumin (BSA) binding was time- and concentration-dependent with hapten densities (i.e., the extent of selective lysine residue modification) and anti-piperacillin antibody binding affinity increasing with increasing molar ratios. Lysine residues in BSA at positions 4, 12, 131, 132, 136, 211, 431, 524, and 537 were modified by piperacillin. Epitope profiles also showed similar lysine residues were modified with amoxicillin, benzylpenicillin and flucloxacillin though the extent of ionisation at each site of modification was drug-dependent. A hapten inhibition ELISA used to assess the specificity of the antidrug antibodies revealed the total antibody binding to aztreonam, amoxycillin, benzylpenicillin and penicillin V BSA adducts. This indicates a lack of cross-reactivity with piperacillin-specific IgG antibodies. Subsequently, LTT and ELISA were employed to screen the piperacillin-specific T cell response and IgG antibodies during piperacillin therapy. It was established that piperacillin-specific T cells were detectable on and following clinical diagnosis of hypersensitivity. Moreover, piperacillin-specific T cell responses were detected in a small number of patients currently classified as drug tolerant. A significant difference in piperacillin-specific IgG was observed when plasma form LTT positive and negative blood samples were compared. LTT positivity was associated with higher levels of piperacillin-specific IgG. Furthermore, a significant decrease in piperacillin-specific IgG was seen 24 h post-desensitisation (graded drug challenge). Piperacillin-specific T cell clones isolated from hypersensitive patients were used to explore the effect of plasma bearing anti-piperacillin IgG on the T cell response. Eleven piperacillin-specific CD4+ and CD8+ T-cell clones were generated from 2 hypersensitive patients. All clones were stimulated to proliferate with piperacillin in a concentration-dependent manner. IFN-γ and IL-5 secretion was seen to predominate following piperacillin stimulation. There were no differences in piperacillin-specific T-cell proliferation when piperacillin-specific antibody bearing plasma and plasma from naive volunteers were compared. However, attenuation in IFN-γ secretion was observed with plasma bearing anti-piperacillin antibodies alone. Collectively, the data presented in this thesis begins to describe the different components of the drug-specific humoral and cellular immune response that develops in piperacillin hypersensitive patients with CF.

In recent years, a number of reports have focused on the use of polyesters in drug delivery due to their intrinsic biocompatibility and biodegradability. In this thesis, aliphatic polyesters were synthesized by polycondensation reaction and ring opening polymerization reactions. The properties of the polymers and drug delivery potential of the resultant materials were evaluated. In the polycondensation reactions, a series of aliphatic polyesters of similar molecular weight were synthesized by reacting 1,10-decanediol with different ratios of succinic acid/phenylsuccinic acid and the effects of phenyl group side-chain substitution on polymer properties was investigated. A solvent-free melt polycondensation method using scandium (III) triflate as catalyst at an industrially relevant temperature (120 °C) was used. As the phenyl content increased, the polymers changed from semicrystalline to amorphous in state. The loading capability of polymers was checked by formulating nanoparticles containing coumarin 6 as a fluorescent dye analogue of active drugs. A polymer with a 70/30 ratio of succinic acid and phenylsuccinic acid showed the highest dye loading among the set of materials synthesised. This polymer was found to be degradable over time under selected experimental conditions. Amphiphilic block co-polymers from the PluronicTM class were used to stabilize, in PBS, nanoparticles formed from these polyesters by nanoprecipitation routes. The metabolic activity, cell membrane integrity and lysosomal functions of C3A cells dosed with the polymers were determined to observe the cytocompatibility of the highest dye-loaded nanoparticles. Activity relative to undosed C3A cells was retained at more than 80% in the all of the assays. Imaging of Pluronic coated and uncoated nanoparticles in C3A cells suggested that both types of the nanoparticles were endocytosed in the early stage of the study (within 10 min). The internalization of nanoparticles was increased progressively over the study time. These results indicated the possible utility of the selected polymers in diagnostic and delivery applications. Ring opening polymerization (ROP) reactions were used for the synthesis of a diblock (mPEG-b-PεDL) and a triblock (PεDL-b-PEG-b-PεDL) copolymer from a seven membered ε-decalactone (ε-DL) monomer obtained from renewable sources. A diblock (mPEG-b-PεDL) copolymer was compared with structurally similar mPEG-b-PCL copolymer synthesized via ROP of ε-caprolactone (ε-CL) monomer, which can be considered as a non-renewable monomer. A six membered δ-decalactone (δ-DL) was also used for the synthesis of a diblock copolymer (mPEG-b-PδDL) to compare the reaction kinetics and properties of the copolymers. The copolymers were prepared via bulk polymerisation using 1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD) as a metal-free catalyst to replace the conventionally used stannous octoate [Sn(Oct)2]. A higher polymerization efficiency was achived with TBD compared to Sn(Oct)2 catalyst. However, a notable difference in the reaction temperature required for ε-DL and δ-DL polymerization was observed. The comparison with a structural analogue, i.e. ε-CL, demonstrated that the ε-DL polymerization was inhibited due to the presence of the alkyl chain of ε-DL monomer. However, a higher reaction time (12 h for TBD and 24 h for Sn(Oct)2) in CROP of ε-DL was addressed by using microwave based ring opening polymerization (MROP) reaction. The MROP was adopted as a ‘green’ and cheap heating method alternative to conventional heating (CROP) for the synthesis of mPEG-b-PεDL diblock copolymers using TBD as a catalyst. All the reactions were conducted in bulk. The MROP was designed based on the dielectric properties of all the reacting materials, as it was found that ε-DL monomers showed good absorption of MW radiation (tanδ>0.5). Accordingly, MROP resulted in a higher rate of ε-DL polymerization compared to CROP but comparison of the synthesis of mPEG-b-PCL copolymer by MROP indicated that the presence of the alkyl chain in ε-DL monomer significantly reduced the rate of polymerization. The synthesized mPEG-b-PεDL copolymer was investigated as a potential drug delivery vehicle for solubilization and controlled delivery of indomethacin. The indomethacin loading and release from mPEG-b-PεDL micelles (amorphous core) was compared against well-established mPEG-b-PCL micelles (semicrystalline core). The drug-polymer compatibility was also determined through a predictive computational approach to access the drug solubilisation (or drug loading) into hydrated micelles. The micelles were prepared by solvent evaporation method and characterized for size, morphology, indomethacin (IND) loading and release. Both of the micelle formulations showed a uniform distribution of spherical micelles with size <60 nm. However, a significantly higher size of empty mPEG-b- PεDL micelle was observed compared to mPEG-b-PCL micelles. A higher compatibility of the drug was predicted with PCL core as determined by modified Flory-Huggins interaction parameters (sp) using the Hanson solubility parameter (HSP) approach. The compatibility of the drug was determined for both of the segments (hydrophilic and hydrophobic) of the copolymers and found to be in the order of sp (PεDL)> sp (mPEG)> sp (PCL). The predictions suggested that more IND should encapsulate within the micelles with PCL core compared to PDL core, but the IND loading experiments revealed an overall higher loading in PεDL core (6.55 wt%) compared to PCL core (5.39 wt%) (P < 0.05, unpaired student’s t-test). However, consideration of the IND loading per unit volume of the micelles revealed that the PCL cored micelles was able to load 1.5 times more compared to the PεDL cored micelles. This result illustrated the higher compatibility of the IND with PCL core in accordance with the solubility parameter calculations. These data also suggested that the overall higher IND loading in PεDL core was attributable to the amorphous nature of the core which increased the core volume by 1.81 times compared to the PCL core. Drug release studies showed the sustained release pattern from both of the micelle systems although the semicrystalline PCL core (80% drug release in 110 h) was able to release the drug for a longer period compared to PεDL core (80% drug release in 72 h). Cell viability tests demonstrated the cytocompatibility of the mPEG-b-PεDL polymer. The micelles were internalized effectively in the early stages of the study and progressively increased with time. The results of the present thesis suggested that novel aliphatic polyester can be good candidates for the drug delivery applications and further studies can explore the possible applications of these polymers in the biomedical field.

The biopharmaceutical market has been on the rise for the past two decades and is expected to continue to excel, currently presenting a growing rate of more than double than conventional pharma. Traditionally this growth has been hindered by multiple formulation issues such as poor bioavailability and poor stability. Consequently, the drive to optimise the stability of protein drug candidates via formulation impels the need for development of novel excipients. Novel glycopolymer excipients were reported to confer improved protein stability in selected cases. Nonetheless,their structure-function relationship and wider applicability remain largely unknown. Here we report the synthesis of glycopolymers with different molecular architectures based on mannose, galactose, arabinose, N-acetyl glucosamine, lactose and trehalose, and nvestigate their utility as excipients for the solution formulation of a monoclonal antibody (mAb). In this thesis work the physical stability of selected antibodies was measured as the unfolding transition temperature (Tm) and aggregation onset temperature (Tagg), as a function of glycopolymer properties, such as the nature of sugar repeating unit, macromolecular architecture and concentration. Results show that, in contrast to the stabilising effect of the corresponding mono- and di-saccharide constituents, both linear and 4-arm star glycopolymers generally destabilised the antibody, decreasing both Tm and Tagg. Accelerated stability studies of a concentrated mAb solution followed the same trend, where an increasing glycopolymer:mAb molar ratio generally decreased the percentage monomer(i.e. increased soluble aggregates). Importantly, trehalose-based glycopolymers further generated visible aggregates that could not be predicted from Tm or Tagg data. The data demonstrate a complex interplay of sugar chemistry and solution concentration of synthetic glycopolymers on their modulation of protein conformational stability and aggregation propensity. The mechanisms involved in protein:glycopolymer interaction, both in solution and dry state were further investigated, thus unravelling the behaviour reported in terms of protein stabilisation. Finally, the glycopolymers were studied as drug delivery systems, acting as solubility enhancers for hydrophobic species in aqueous solutions, through the use of extrinsic fluorescent dyes.

The mupirocin biosynthetic pathway belongs to the trans-AT group in which acyltransferase (AT) activity is provided by a separate polypeptide (MmpC) rather than in cis as found in the typical type I polyketide synthases. AT docking domains have been documented in trans-AT PKS clusters for ten years yet little functional evidence is available. The cluster shows many interesting features that must be understood to create novel products. Specificity studies demonstrated that AT2 performs the typical AT function of loading malonyl-CoA to ACPs throughout the cluster. Mutagenesis studies demonstrated the importance of AT active site residues for protein structural integrity, acquisition and transfer of malonate and propose an alternate role for AT1 as a proofreading enzyme responsible for hydrolysing truncated intermediates from the pathway. Consequently an edit, reload, reduce model for MmpC is proposed. Mutagenesis of docking domains led to a halt in mupirocin production and suggested that docking domains are required for structural integrity of the Mmps or for guiding the ACPs into the correct position for interactions with their respective partners. Studies involving a mutated ACP3 protein confirmed the importance of Trp55, as demonstrated by structural changes and the inability of the protein to accept malonate from AT2.

Graphene oxide (GO) and reduced GO (r-GO) nanomaterials exhibit great potential for several biomedical applications. Of foremost importance is to determine any potential health hazards related in their exposure. In this research, we hypothesised that the different material properties evidenced by GO and r-GO would elicit different biological responses. The first objective of this work was to synthesize Go and r-GO and characterize their physiochemical properties. The second aim was to investigate whether the two-distinct surface chemistries of GO and r-GO influenced their biological effect. The potential toxicity of these nanomaterials was investigated using the normal lung fibroplast cell line MRC-5 and cancerous epithelial lung cell line A549. The cytotoxicity of graphene derivatives was concentration-, time- and cell-dependent and varied according to the material used. Thus, the surface chemistry of graphene plays a critical role in its biocompatibility. Non-cancerous cells had a higher sensitivity to GO cytotoxicity than cancer cells. R-GO was highly biocompatible to MRC-5 cells and for A549 cells had a minimal effect of cell viability. At 37C˚, GO and r-GO were moderately hemolyric at concentration of 125 µg/ml and highly haemolytic at concentration of 300 µg/ml. Exposure of cells to both graphene derivatives led to reactive oxygen species (RO5) generation without genotoxicity. GO, but not r-GO, led to autophagy in both cell lines, possibly inhibiting the PIP3-Akt/mTOR pathway. For both cell lines and at non-lethal concentrations, GO downregulated the expression of glycogen synthase kinase-3 (GSK-3ß). GO was also found to dysregulate both Wnt/b-catenin and Akt cell signalling pathways which are vital for cellular function. The finding relating to cell signalling provide an insight to the safety of GO which is important to its use in cancer therapy.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, leading to hepatic steatosis, fibrosis, cirrhosis and hepatocellular carcinoma. A vaccine is currently not available, while the standard of care is effective in only 50% of treated patients. The first specific anti-HCV drugs have been recently approved, and new classes of targeted agents are under clinical trials/investigation. Nevertheless, improved treatment strategies are needed, in order to bypass the rapid emergence of resistance. All the viral non-structural proteins are a possible target for the identification of novel and selective antivirals. Among them, the NS3 helicase is still underexploited, with no known inhibitor under pre-clinical or clinical development. This enzyme plays a crucial role in the virus life cycle: it catalyses the separation of double-stranded RNA strands, which is necessary for genome amplification and translation. Due to its essential function, the NS3 helicase was chosen as a target for the identification of new, specific anti-HCV compounds. Different computer-aided techniques were employed to identify potential smallmolecule inhibitors of the enzyme. Two structure-based virtual screenings of commercially available compounds were performed on the main nucleic acid binding site. A series of candidate inhibitors was evaluated in the HCV replicon assay, yielding two primary hits with low μM activity. Secondly, the model of the one known inhibitor co-crystallised with the enzyme was used as a starting point for a shape-comparison screening of small molecule libraries. A new series of compounds was selected and evaluated for anti-HCV activity, and one of them was found to inhibit the viral replication at a low μM concentration. Several new derivatives of the initial hits were synthesised, belonging to four main structural families: bis-aromatic piperazine derivatives, symmetrical phenylendiamine compounds, differently substituted thieno-pyrimidines, and triphenyl-pyrrolone analogues. Inhibition of HCV replication in the replicon assay was evaluated for the new compounds prepared and several structures showed a range of activity from low-μM to nM.

The successful clinical translation of non-viral gene delivery systems has yet to be achieved due to the biological and technical obstacles to preparing a safe, potent and cost-effective vector. Hyperbranched polymers have emerged as promising candidates to address gene delivery barriers owing to their relatively simple synthesis and ease of modification compared to other polymers, which makes them more feasible for scale up and manufacturing. In the first part of this thesis, we compare hyperbranched poly(amino acids) synthesised by co-polymerising histidine and lysine, with hyperbranched polylysine prepared using the well-known 'ultra-facile' thermal polycondensation route, to investigate the effects of histidine units on the structure and gene delivery applications of the resultant materials. The conditions of polymerisation were optimised to afford water-soluble hyperbranched polylysine-co-histidine of three different molar ratios with molecular masses varying from 13-30 kDa. Spectroscopic, rheological and thermal analysis indicated that the incorporation of histidine modulated the structure of hyperbranched polylysine to produce a more dendritic polymer with less flexible branches. Experiments to probe gene delivery to A549 and H1299 cells, surprisingly, indicated that the co-polymers containing histidine were not more effective in transfecting a luciferase gene than hyperbranched polylysines synthesised as established literature comparators. We attribute the variations in gene delivery efficacy to the changes induced in polymer architecture by the branching points at histidine residues, and obtain structure-function information relating histidine content with polymer Tg, pKa and ability to form stable polyplexes with plasmid DNA. These results are of significance to nanomedicine design as they indicate that addition of histidine as a co-monomer in the synthetic route to hyperbranched polymers changes not only the buffering capacity of the polymer but has significant effects on the overall structure, architecture and gene delivery efficacy. It has become known that many cationic polymers are cytotoxic and although a large number of polycations have now designed to address the toxicity problem, there is still a practical need to develop a fast and reliable method for assessing the safety of these materials. In this regard, metabolomics provides a high throughput and comprehensive method that can assess the potential toxicity at the cellular and molecular level. Therefore, in the second part of this thesis, metabolomics was applied to investigate the impact of hyperbranched polylysine, hyperbranched polylysine-co-histidine and branched polyethylenimine polyplexes, on the metabolic pathways of A459 and H1299 cell lines. The study revealed that the polyplexes downregulated metabolites associated with glycolysis and the TCA cycle, and induced oxidative stress in both cell lines. The fold changes of the metabolites indicated that the polyplexes of polyethylenimine and hyperbranched polylysine affected the metabolism much more than the polyplexes of hyperbranched polylysine-co-histidine. This was in line with transfection results, suggesting a correlation between the toxicity and transfection efficiency of these polyplexes. This part highlights the importance of metabolomics approaches not just to assess the potential toxicity of polyplexes but also to understand the molecular mechanisms underlying their action, which could help to design more efficient vectors. In the third part of this thesis, we investigated the ability of the hyperbranched polymers to condense and deliver siRNA. The results indicated that the higher molecular mass polymers achieved better siRNA delivery and gene silencing than the lower molecular mass form of the polymers and the lysine-only polymer was more efficient than the histidinylated one. These results can be attributed to the low charge (molecular mass) and stiffness of siRNA molecules in comparison with plasmid DNA, which in combination with the impact of histidine incorporation on the structure of the hyperbranched polymers can also explain the lower efficiency of histidinylated polymers. Overall, this thesis is highlighted the impacts of structural factors on the gene delivery applications of hyperbranched polymers and the importance of these factors to inform the design of new polymeric vectors. Also, metabolomics approaches were introduced to this area, not only to evaluate the safety of gene vectors but also to understand the molecular basis by which these vectors act. The data together suggest that the hyperbranched polymers prepared during thermal polycondensation of amino acids have some efficacy in preliminary gene delivery applications, and that these might be improved with future studies to be a candidate for clinical purposes.