Impact of the physical-chemical properties of poly(lactic acid)-poly(ethylene glycol) polymeric nanoparticles on biodistribution.

Nanoparticle (NP) formulations are inherently polydisperse making their structural characterization and justification of specifications complex. It is essential, however, to gain an understanding of the physico-chemical properties that drive performance in vivo. To elucidate these properties, drug-containing poly(lactic acid) (PLA)-poly(ethylene glycol) (PEG) block polymeric NP formulations (or PNPs) were sub-divided into discrete size fractions and analyzed using a combination of advanced techniques, namely cryogenic transmission electron microscopy, small-angle neutron and X-ray scattering, nuclear magnetic resonance, and hard-energy X-ray photoelectron spectroscopy. Together, these techniques revealed a uniquely detailed picture of PNP size, surface structure, internal molecular architecture and the preferred site(s) of incorporation of the hydrophobic drug, AZD5991, properties which cannot be accessed via conventional characterization methodologies. Within the PNP size distribution, it was shown that the smallest PNPs contained significantly less drug than their larger sized counterparts, reducing overall drug loading, while PNP molecular architecture was critical in understanding the nature of in vitro drug release. The effect of PNP size and structure on drug biodistribution was determined by administrating selected PNP size fractions to mice, with the smaller sized NP fractions increasing the total drug-plasma concentration area under the curve and reducing drug concentrations in liver and spleen, due to greater avoidance of the reticuloendothelial system. In contrast, administration of unfractionated PNPs, containing a large population of NPs with extremely low drug load, did not significantly impact the drug's pharmacokinetic behavior - a significant result for nanomedicine development where a uniform formulation is usually an important driver. We also demonstrate how, in this study, it is not practicable to validate the bioanalytical methodology for drug released in vivo due to the NP formulation properties, a process which is applicable for most small molecule-releasing nanomedicines. In conclusion, this work details a strategy for determining the effect of formulation variability on in vivo performance, thereby informing the translation of PNPs, and other NPs, from the laboratory to the clinic.

Quaternary Ammonium Palmitoyl Glycol Chitosan (GCPQ) Loaded with Platinum-Based Anticancer Agents-A Novel Polymer Formulation for Anticancer Therapy.

Quaternary ammonium palmitoyl glycol chitosan (GCPQ) has already shown beneficial drug delivery properties and has been studied as a carrier for anticancer agents. Consequently, we synthesised cytotoxic platinum(IV) conjugates of cisplatin, carboplatin and oxaliplatin by coupling via amide bonds to five GCPQ polymers differing in their degree of palmitoylation and quaternisation. The conjugates were characterised by 1H and 195Pt NMR spectroscopy as well as inductively coupled plasma mass spectrometry (ICP-MS), the latter to determine the amount of platinum(IV) units per GCPQ polymer. Cytotoxicity was evaluated by the MTT assay in three human cancer cell lines (A549, non-small-cell lung carcinoma; CH1/PA-1, ovarian teratocarcinoma; SW480, colon adenocarcinoma). All conjugates displayed a high increase in their cytotoxic activity by factors of up to 286 times compared to their corresponding platinum(IV) complexes and mostly outperformed the respective platinum(II) counterparts by factors of up to 20 times, also taking into account the respective loading of platinum(IV) units per GCPQ polymer. Finally, a biodistribution experiment was performed with an oxaliplatin-based GCPQ conjugate in non-tumour-bearing BALB/c mice revealing an increased accumulation in lung tissue. These findings open promising opportunities for further tumouricidal activity studies especially focusing on lung tissue.

Brain Gene Silencing with Cationic Amino-Capped Poly(ethylene glycol) Polyplexes.

Therapeutic gene silencing in the brain is usually achieved using highly invasive intracranial administration methods and/or comparatively toxic vectors. In this work, we use a relatively biocompatible vector: poly(ethylene glycol) star-shaped polymer capped with amine groups (4APPA) via the nose to brain route. 4APPA complexes anti- itchy E3 ubiquitin protein ligase (anti-ITCH) siRNA to form positively charged (zeta potential +15 ± 5 mV) 150 nm nanoparticles. The siRNA-4APPA polyplexes demonstrated low cellular toxicity (IC50 = 13.92 ± 6 mg mL-1) in the A431 cell line and were three orders of magnitude less toxic than Lipofectamine 2000 (IC50 = 0.033 ± 0.04 mg mL-1) in this cell line. Cell association and uptake of fluorescently labelled siRNA bound to siRNA-4APPA nanoparticles was demonstrated using fluorescent activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), respectively. Gene silencing of the ITCH gene was observed in vitro in the A431 cell line (65% down regulation when compared to the use of anti-ITCH siRNA alone). On intranasal dosing with fluorescently labelled siRNA-4APPA polyplexes, fluorescence was seen in the cells of the olfactory bulb, cerebral cortex and mid-brain regions. Finally, down regulation of ITCH was seen in the brain cells (54 ± 13% ITCH remaining compared to untreated controls) in a healthy rat model, following intranasal dosing of siRNA-4APPA nanoparticles (0.15 mg kg-1 siRNA twice daily for 3 days). Gene silencing in the brain may be achieved by intranasal administration of siRNA- poly(ethylene glycol) based polyplexes.

The influence of linkages between 1-hydroxy-2(1H)-pyridinone coordinating groups and a tris(2-aminoethyl)amine core in a novel series of synthetic hexadentate iron(III) chelators on antimicrobial activity.

Resistance of pathogens to antimicrobials is a major current healthcare concern. In a series of linked studies, we have investigated synthetic iron chelators based on hydroxy-pyridinone ligands as novel bacteriostatic agents. Herein we describe our synthesis of several useful building blocks based on the 1-hydroxy-2(1H)-pyridinone moiety, including a novel formyl derivative, which were combined with a tris(2-aminoethyl)amine core to obtain a series of new high-affinity hexadentate Fe(III) chelators. The design principle examined by this series is the size and flexibility of the linker between the core and the metal ligands. Measurement of the pKa and stability constants (Fe3+ and Cu2+) of representative coordinating groups was performed to help rationalise the biological activity of the chelators. The novel chelators were tested on a panel of representative microorganisms with some effectively inhibiting microbial growth. We demonstrate that the nature and position of the linker between the hydroxypyridinone and the tris(2-aminoethyl)amine core has considerable impact upon microbial growth inhibition and that both amide or amine linkages can give efficacious chelators.

Limiting the level of tertiary amines on polyamines leads to biocompatible nucleic acid vectors.

We have designed an efficient, synthetic nucleic acid vector, which is relatively non-toxic. [N-(2-ethylamino)-6-O-glycolchitosan - EAGC] polymers were 10-50 fold less toxic than Lipofectamine 2000, able to complex DNA, mRNA and siRNA into positively charged (zeta potential=+40 - 50mV), 50-450nm nanoparticles. The level of tertiary amine N-2-ethylamino substitution (DStert) was inversely proportional to the IC50 of the EAGC polymers in the A431 cell line: IC50=6.18DStert-0.9, r2=0.9991. EAGC polyplexes were stable against a heparin challenge, able to protect the nucleic acids from nuclease degradation and achieve levels of transfection comparable to Lipofectamine 2000 formulations. The relative biocompatibility of the vector allowed 10 fold higher doses of DNA (1µg compared to 0.1µg per well with Lipofectamine 2000) and siRNA (10.7µg per well vs 1.3µg with Lipofectamine 2000) to be applied to cells, when compared to Lipofectamine 2000. Finally intranasal application of EAGC - siRNA complexes resulted in siRNA transfer to the neurons of the olfactory bulb.

Synthesis of novel Iron(III) chelators based on triaza macrocycle backbone and 1-hydroxy-2(H)-pyridin-2-one coordinating groups and their evaluation as antimicrobial agents.

Several novel chelators based on 1-hydroxy-2(1H)-pyridinone coordinating groups decorating a triaza macrocyclic backbone scaffold were synthesised as potential powerful Fe(3+) chelators capable of competing with bacterial siderophores. In particular, a novel chloromethyl derivative of 1-hydroxy-2(1H)-pyridinone exploiting a novel protective group for this family of coordinating groups was developed. These are the first examples of hexadentate chelators based on 1-hydroxy-2(1H)-pyridinone to be shown to have a biostatic activity against a range of pathogenic bacteria. Their efficacy as biostatic agents was assessed revealing that minor variations in the structure of the chelator can affect efficacy profoundly. The minimal inhibitory concentrations of our best tested novel chelators approach or are comparable to those for 1,4,7-tris(3-hydroxy-6-methyl-2-pyridylmethyl)-1,4,7-triazacyclononane, the best Fe(3+) chelator known to date. The retarding effect these chelators have on microbial growth suggests that they could have a potential application as a co-active alongside antibiotics in the fight against infections.