(T1330-01-03) Lipid Conjugation to Brush PEG Polymer Delivery Systems Enables Hitchhiking on Lipid Trafficking Pathways to Manipulate Plasma Half-Life, Lymph Uptake and Biodistribution

Purpose: Compared with conventional drug formulations, drug carriers can provide many advantages such as improved water solubility, increasing drug payload and targeted delivery and release in specific tissues [1, 2]. Recently, lipidation of drug carriers as well as peptide therapeutics, vaccines and adjuvants has been utilised to prolong elimination half-life [3], and increase lymph node uptake following subcutaneous dosing [4]. These beneficial changes to pharmacokinetics (PK) and biodistribution have been proposed to result from association with and ‘hitchhiking’ on endogenous lipid trafficking pathways. In this project, we conjugated a range of different lipids to novel brush polyethylene (PEG) polymers for the first time to determine impact on tissue biodistribution, plasma PK and lymph uptake. We also investigated the mechanisms driving changes to PK and biodistribution, including binding to serum albumin as well as lipoproteins.

Methods: Brush PEG polymers were synthesised by copper-mediated polymerisation. The different lipids attached to the PEG polymers included a single short C2 hydrocarbon tail (1C2-PEG (molecular weight: 5636 Da)), single medium-chain C12 hydrocarbon tail (1C12-PEG (molecular weight: 5776 Da)), diacylglycerol moiety containing two medium-chain C12 hydrocarbon tails (2C12-PEG (molecular weight: 6046 Da)), diacylglycerol moiety containing two long-chain C18 fatty acids (2C18-PEG (molecular weight: 6131 Da)) and cholesterol (Cho-PEG (molecular weight: 5977 Da)). The polymers were also attached to Cyanine 5 dye for imaging and quantification purposes. A single-run density gradient ultracentrifugation-based assay was used to evaluate in vitro association of the polymers with bovine (BSA) and rat serum albumin (RSA), and also protein and lipoprotein fractions in lymph and plasma from fed and fasted rats. In vivo plasma PK and thoracic lymph uptake of the polymers was assessed in Sprague-Dawley rats following intravenous (IV) and subcutaneous (SC) administration to carotid artery and/or thoracic lymph cannulated rats. Tissue biodistribution was assessed in C57BL/6 mice following SC administration. The concentration of polymers in plasma and lymph was quantified by a fluorescence plate reader. The distribution of the polymers into major organs and tissues was analysed using an in vivo imaging system. Data are presented as mean ± standard deviation (SD). One-way or two-way ANOVAs were used to compare groups when there were one or two independent variables, respectively. Tukey’s post-hoc test was performed to detect any statistical difference between specific groups. A P value ≤ 0.05 was considered statistically significant.

Results: 2C12-PEG, Cho-PEG and 2C18-PEG polymers had statistically longer plasma half-life (10.7 ± 0.4, 13.0 ± 2.5 and 12.0 ± 3.0 hours, respectively) than the other polymers (2.6 ± 0.4 hours for 1C2-PEG and 3.3 ± 1.6 hours for 1C12-PEG) following IV dosing. A SC bioavailability of > 20% was observed for all brush PEG polymers. Interestingly, the elimination half-life of the 2C12-PEG and Cho-PEG polymers following SC dosing was statistically longer than the elimination half-life of the same polymers following IV dosing (22.1 ± 5.4 vs 13.0 ± 2.5 for Cho-PEG (P < 0.05), and 17.0 ± 1.3 vs 10.7 ± 0.4 (P < 0.05) for 2C12-PEG), which indicates a flip-flop PK behaviour for the 2C12-PEG and Cho-PEG polymers following SC administration. 2C18-PEG had the highest plasma exposure (determined by area under the plasma concentration-time curve) following IV and SC dosing (Fig. 1A and 1B). The proportion of the bioavailable dose that was lymphatically transported was statistically higher for 2C12-PEG polymer (28% ± 5.3) than other polymers (Fig. 1C and 1D). Unexpectedly, 1C2-PEG had statistically higher lymph transport compared to Cho-PEG and 1C12-PEG (Fig. 1C), which might be explained by the surprisingly higher association of this polymer with low-density lipoproteins. The polymers had distinct distribution patterns into major organs and several tissues (Fig. 2). Finally, the polymers had different association with lipid trafficking pathways. For example, 2C12-PEG had the highest association with BSA and RSA (Fig. 3A and 3B). The different association between the polymers with endogenous lipid trafficking pathways (mainly albumin and lipoproteins) might explain the variation in the polymers’ in vivo distribution.

Conclusion: Lipid functionalisation of brush PEG polymers influences their association with endogenous lipid trafficking pathways, plasma PK, lymph uptake and biodistribution. Plasma half-life and plasma exposure was enhanced for 2C12-PEG, Cho-PEG and 2C18-PEG following IV and SC administration compared to 1C2-PEG and 1C12-PEG polymers. The distinct biodistribution characteristics of each of these polymers suggests that the design of the polymers may be chosen to achieve a desired profile depending on the therapeutic application. For instance, lymph uptake following SC dosing was highest for 2C12-PEG and unexpectedly 1C2-PEG, probably because of their association with specific lipoproteins. This makes these two polymers potential drug carriers for targeting the lymphatic system to treat or diagnose a wide list of diseases involving the lymphatic system such as many infectious diseases (e.g. HIV, hepatitis B), autoimmune diseases, cancers and acute diseases [5].

References: 1) Patra, Jayanta Kumar, et al. "Nano based drug delivery systems: recent developments and future prospects." Journal of nanobiotechnology 16.1 (2018): 1-33.
2) Ross, Kathleen A., et al. "Nano-enabled delivery of diverse payloads across complex biological barriers." Journal of Controlled Release 219 (2015): 548-559.
3) Frich, Camilla Kaas, et al. "Non-covalent hitchhiking on endogenous carriers as a protraction mechanism for antiviral macromolecular prodrugs." Journal of controlled release 294 (2019): 298-310.
4) De Vrieze, Jana, et al. "Lipid Nature and Alkyl Length Influence Lymph Node Accumulation of Lipid‐Polyethylene Glycol Amphiphiles." Advanced Therapeutics 4.8 (2021): 2100079.
5) Abdallah, Mohammad, et al. "Lymphatic targeting by albumin-hitchhiking: applications and optimisation." Journal of Controlled Release 327 (2020): 117-128.

Acknowledgments: This work was funded by Australian Research Council DP220103654.