Using glycosaminoglycan/chemokine interactions for the long-term delivery of 5P12-RANTES in HIV prevention - PubMed (original) (raw)

. 2013 Oct 7;10(10):3564-73.

doi: 10.1021/mp3007242. Epub 2013 Aug 26.

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Using glycosaminoglycan/chemokine interactions for the long-term delivery of 5P12-RANTES in HIV prevention

Nick X Wang et al. Mol Pharm. 2013.

Abstract

5P12-RANTES is a recently developed chemokine analogue that has shown high level protection from SHIV infection in macaques. However, the feasibility of using 5P12-RANTES as a long-term HIV prevention agent has not been explored partially due to the lack of available delivery devices that can easily be modified for long-term release profiles. Glycosaminoglycans (GAGs) have been known for their affinity for various cytokines and chemokines, including native RANTES, or CCL5. In this work, we investigated used of GAGs in generating a chemokine drug delivery device. Initial studies used surface plasmon resonance analysis to characterize and compare the affinities of different GAGs to 5P12-RANTES. These different GAGs were then incorporated into drug delivery polymeric hydrogels to engineer sustained release of the chemokines. In vitro release studies of 5P12-RANTES from the resulting polymers were performed, and we found that 5P12-RANTES release from these polymers can be controlled by the amount and type of GAG incorporated. Polymer disks containing GAGs with stronger affinity to 5P12-RANTES resulted in more sustained and longer term release than did polymer disks containing GAGs with weaker 5P12-RANTES affinity. Similar trends were observed by varying the amount of GAGs incorporated into the delivery system. 5P12-RANTES released from these polymers demonstrated good levels of CCR5 blocking, retaining activity even after 30 days of incubation.

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Figures

Figure 1

Figure 1

SPR analysis of GAGs (heparin, CSA and CSB) interactions with immobilized 5P12-RANTES. Overlayed sensorgrams show initial background wash (1min), followed by injection of GAG solution and association of GAGs with the immobilized 5P12-RANTES on the chip surface over the following 1.5min, and then subsequent washing (dissociation) of GAGs from the surface for the remaining 4.5min.

Figure 2

Figure 2

SPR concentration analysis of heparin/5P12-RANTES interactions. Overlayed sensorgrams showing initial background wash (1min), followed by injection of heparin at 0.25, 0.5, 1.0, 2.5, 5.0, 10 and 20 µM for the next 1.5min, and then subsequent washing (dissociation) of heparin from the surface for the remaining 4.5min.

Figure 3

Figure 3

SPR concentration analysis of CSB/5P12-RANTES interactions. Overlayed sensorgrams showing initial background wash (1min), followed by injection of heparin at 0.25, 0.5, 1.0, 2.5, 5.0, 10 and 20 µM for the next 1.5mins, and then subsequent washing (dissociation) of CSB from the surface for the remaining 4.5min.

Figure 4

Figure 4

FTIR of BSA, heparin, heparin mixed with BSA (no conjugation) and crosslinked heparin/BSA. The spectra of crosslinked BSA and heparin occurred after multiple, extensive washings. The presence of both BSA and heparin confirm that crosslinking occurred. Small changes in OH groups indicate that conjugation could be through coupling to the GAG carboxylate.

Figure 5

Figure 5

5P12-RANTES released at each time point from polymers containing different GAGs. Heparin/BSA (○) disks showed the highest level of sustained release. CSB/BSA (formula image) disks also showed substantial and sustained levels of release. CSA/BSA (♦) disks resulted in release profiles with the lowest sustained release, similar to the release in BSA-only control disks (not shown). Error bars represent standard deviation of means.

Figure 6

Figure 6

Normalized cumulative release profile from GAG/BSA polymers. The Heparin/BSA(○) disks resulted in the most sustained release, followed by CSB/BSA(formula image) disks. The CSA/BSA(♦) disks resulted in the least sustained release. Error bars represent standard deviation of means. The main graph and the insert presented in the figure represent the same data. The main graph is zoomed in to highlight the sustained release.

Figure 7

Figure 7

5P12-RANTES release from polymers containing incremental heparin fractions - BSA only (no heparin) (♦), 2.5% heparin (formula image), 5% heparin (formula image), 15% heparin (×) and 25% heparin (formula image). All the release curves are characterized by an initial burst phase, and then followed by a sustained release. In the burst phase, an increase in heparin content appears to decrease the burst effect, whereas in the sustained release, increases in heparin content corresponded with increases in release at each time point. Error bars represent standard deviation of means.

Figure 8

Figure 8

Normalized cumulative release profiles from heparin/BSA polymers. Gels tested include: BSA only (no heparin) (♦), 2.5% heparin (formula image), 5% heparin (formula image), 15% heparin (×) and 25% heparin (formula image). The main graph and the insert presented in the figure represent the same data. The main graph is zoomed in to highlight the sustained release. Sustained release from the heparin/BSA disks corresponded with heparin content, more sustained release profiles were observed for polymers with higher heparin content. Error bars represent standard deviation of means.

Figure 9

Figure 9

CCR5 blocking capacity of stock 5P12-RANTES as determined by monoclonal antibody (clone 2D7) binding. In samples where 5P12-RANTES concentrations were greater than 100ng/ml, 2D7% presentation was less than 1%, indicating good CCR5 blocking. In samples where 5P12-RANTES concentrations were below 0.8ng/ml, 2D7% presentation was at or greater than 20%, suggesting little to no CCR5 blocking (this level is similar to that of no 5P12-RANTES, the negative control). Insets show FACS histograms of 2D7 presentation on the studied PBMCs at low and high 5P12-RANTES concentrations.

Figure 10

Figure 10

CCR5 blocking activity of the released aliquots from GAG/BSA polymers. Release aliquots from BSA and 25% heparin disks at 3 time points (6h, 265h and 650h) were evaluated. Protein concentration were previously determined using ELISA, and samples were diluted to concentrations both one order of magnitude above and below the CCR5 blocking threshold. Each symbol represents that release sample at all of its tested dilution concentrations. Release samples at or above the blocking threshold concentration showed good blocking, while samples diluted to below the blocking threshold concentration showed poor blocking, comparable to that of stock 5P12-RANTES.

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