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Comment
. 2011 Oct;4(5):631-43.
doi: 10.1007/s12265-011-9292-0. Epub 2011 Aug 11.

N-acetylglucosamine conjugated to nanoparticles enhances myocyte uptake and improves delivery of a small molecule p38 inhibitor for post-infarct healing

Warren D Gray  1 , Paolin Che, Milton Brown, Xinghai Ning, Niren Murthy, Michael E Davis
Affiliations
Comment

N-acetylglucosamine conjugated to nanoparticles enhances myocyte uptake and improves delivery of a small molecule p38 inhibitor for post-infarct healing

Warren D Gray et al. J Cardiovasc Transl Res. 2011 Oct.

Abstract

An estimated 985,000 new myocardial infarctions will occur in the USA in 2011. While many will survive the initial insult, the early damage will eventually lead to heart failure for which the only definitive cure is transplantation. Cardiomyocyte (CM) apoptosis is a large contributor to cardiac dysfunction, and although potential therapeutic molecules exist to inhibit apoptotic pathways, drug delivery methods are lacking. This damage is largely regional and thus localized delivery of therapeutics holds great potential; however, CMs are relatively non-phagocytic, which limits existing options that rely on phagocytosis. Recently, the sugar N-acetylglucosamine (GlcNAc) was shown to be bound and internalized by CMs, providing a potential mechanism for drug delivery. Here we demonstrate efficacy of a drug delivery system comprising a drug-loaded biodegradable polyketal nanoparticle that is surface-decorated with GlcNAc. Inclusion of the sugar enhanced uptake by CMs as measured by intracellular activated fluorescence. When delivered in vivo following ischemia-reperfusion injury, GlcNAc-decorated particles loaded with the p38 inhibitor SB239063 reduced apoptotic events and infarct size and improved acute cardiac function. This was in contrast to our published data demonstrating no acute effect of non-sugar-decorated, p38 inhibitor-loaded particles. These data suggest a novel therapeutic option to enhance uptake of drug-loaded nanoparticles to CMs and perhaps reduce the large amount of CM cell death following myocardial injury.

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Conflict of interest statement

Conflict of interest statement

Drs. Davis and Murthy, as well as Emory University, are entitled to equity and royalties derived from Ketal Biomedical Incorporated, which is developing products related to the technology described in this paper. This study could affect his/her/their personal financial status. The terms of this arrangement have been reviewed and approved by Emory University in accordance with its conflict of interest policies.

Figures

Figure 1
Figure 1. Overall scheme for proposed GlcNAc-mediated drug delivery system
(a) Decoration of drug-loaded polymeric nanoparticle with GlcNAc should provide a mechanism whereby therapeutics may be delivered intracellularly to CMs. (b) The particle will be composed of the acid-labile poly(cyclohexane-1,4-diyl acetone dimethylene ketal) (PCADK). Once internalized, the particle will degrade into the biocompatible products acetone and cyclohexanedimethanol.
Figure 2
Figure 2. Synthesis of GlcNAc-alkyl
A facile and scalable 8-step process is followed to produce the tethering and targeting compound GlcNAc-alkyl. During nanoparticle production, the hydrophobic alkyl chain associates with the polymeric particle and the hydrophilic carbohydrate headgroup is free to interact with CMs.
Figure 3
Figure 3. Incubation of CMs with PK-GlcNAc-rhodamine particles for internalization verification
(a) Rhodamine-loaded particles (diameter=320±156 nm) were decorated with GlcNac-alkyl and imaged by SEM. (b) Cultured CMs were treated with PK-GlcNAc-rhodamine particles for 12h before fixation in paraformaldehyde and immunostaining for CM-specific α-actinin (green) and DAPI for nuclei (blue). Confocal microscopy afforded orthogonal views of the cell, by which internalized nanoparticles (red) can be observed in the same plane as the α-actinin (arrows).
Figure 4
Figure 4. Measurement of particle internalization by CMFDA fluorescence demonstrating increased uptake
PK-GlcNAc-CMFDA nanoparticles were decorated with 0%, 0.6%, or 9% GlcNAc-alkyl by weight and loaded with the cell tracker dye, 5-chloromethylfluorescein diacetate (CMFDA). (a) Particles are closely load-matched for CMFDA content: 0%, 0.6% and 9% decorated particles contained 2.9±0.3, 5.1±1.5, and 4.3±0.2 nmol CMFDA/mg particle, respectively. Data are expressed as mean±SEM from three experiments. (b) After incubation of CMs with particles, fluorescence of cell culture was obtained by plate reader and normalized to 0% GlcNAc-alkyl decoration and respective particle CMFDA content. The positive correlation between fluorescence and degree of decoration indicates a dose response and confirms cell uptake of GlcNAc-decorated particles. Data are expressed as mean±SEM from four separate experiments (*p<0.05, ***p<0.001; ANOVA followed by Newman-Keuls post-test).
Figure 5
Figure 5. Treatment of CMs in vitro with p38 inhibitor-loaded nanoparticles reduces TNF-α stimulated p38 activation
PK-GlcNAc and PK-GlcNAc-SB particles were imaged via (a) SEM (diameter=407±125 nm or d=342±165 nm, respectively) and (b) analyzed by DLS (diameter=465±173 nm or diameter=395±145 nm, respectively). Data are mean±SD. (c) PK-GlcNAc-SB particles release cargo through diffusion at pH 7.4. (d) PK-GlcNAc did not prevent p38 phosphorylation, as demonstrated by a 2.4-fold increase of p-p38 due to TNF-α treatment. However, PK-GlcNAc-SB treatment significantly inhibited p38 activation compared with empty particles. Data are mean±SEM and are expressed as a ratio of phosphorylated to total p38 (n=3; *p<0.05; ANOVA followed by Tukey post-test.)
Figure 6
Figure 6. GlcNAc decoration enhances particle uptake in vivo following IR and reduces apoptotic events
(a) Rats that received myocardial injection of (9′-(4-(and 5)-chloromethyl-2-carboxyphenyl)-7′-chloro-6′-oxo-1,2,2,4-tetramethyl-1,2-dihydropyrido[2′,3′-6]xanthene (CMRA)-loaded GlcNAc-decorated particles (PK-GlcNAc-CMRA) immediately following IR exhibited a greater CMRA fluorescence three days post-IR than animals that had received PK-CMRA particles, indicating enhanced in vivo uptake due to GlcNAc decoration. (b and c) Immediately following IR, particles were injected intramyocardially and hearts removed one day post-IR. TUNEL staining indicated apoptotic nuclei (b) (apoptotic nuclei [purple] indicated by arrow, non-apoptotic nuclui [blue] indicated by triangle), and comparison against total cardiomyocyte nuclei indicated that animals that received PK-GlcNAc-SB particle injections achieved 3.6-fold fewer apoptotic events in the injured myocardium compared to IR and 2.7-fold fewer than the PK-GlcNAc group (c). (n=3 for each group). Data are mean±SEM. *p<0.05, *p<0.01. ANOVA followed by Newman-Keuls post-test.
Figure 7
Figure 7. Infarct size reduction and functional improvements seen in PK-GlcNAc-SB treatment following ischemia-reperfusion injury
Immediately following IR, particles were injected directly into the injured myocardium. Three days following surgery, echocardiography was performed and heart cross sections analyzed for infarct size. (a) The ratio of infarct size to area at risk was calculated for the treatment groups. PK-GlcNAc treatment had no significant effect, but PK-GlcNAc-SB treatment significantly decreased infarct size compared to both treatment groups (mean±SEM, n=5; *p<0.05, **p<0.01; ANOVA followed by Newman-Keul multiple comparison post-test). (b) Fractional shortening calculated from echocardiographic measurements demonstrated a significant decrease in function in IR animals compared to sham. There was no significant improvement seen with PK-GlcNAc, though PK-GlcNAc-SB treatment improved function compared with other IR groups. (n=4 for sham n>7 for IR groups). Data are mean±SEM. *p<0.05, ***p<0.001. ANOVA followed by Tukey post-test.
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