Carbohydrate polymers for nonviral nucleic acid delivery

Abstract

Carbohydrates have been investigated and developed as delivery vehicles for shuttling nucleic acids into cells. In this review, we present the state of the art in carbohydrate-based polymeric vehicles for nucleic acid delivery, with the focus on the recent successes in preclinical models, both in vitro and in vivo. Polymeric scaffolds based on the natural polysaccharides chitosan, hyaluronan, pullulan, dextran, and schizophyllan each have unique properties and potential for modification, and these results are discussed with the focus on facile synthetic routes and favorable performance in biological systems. Many of these carbohydrates have been used to develop alternative types of biomaterials for nucleic acid delivery to typical polyplexes, and these novel materials are discussed. Also presented are polymeric vehicles that incorporate copolymerized carbohydrates into polymer backbones based on polyethylenimine and polylysine and their effect on transfection and biocompatibility. Unique scaffolds, such as clusters and polymers based on cyclodextrin (CD), are also discussed, with the focus on recent successes in vivo and in the clinic. These results are presented with the emphasis on the role of carbohydrate and charge on transfection. Use of carbohydrates as molecular recognition ligands for cell-type specific delivery is also briefly reviewed. We contend that carbohydrates have contributed significantly to progress in the field of non-viral DNA delivery, and these new discoveries are impactful for developing new vehicles and materials for treatment of human disease.

Fig. 1

(a) Synthesis of dextran-spermine conjugates. (b) Fluorescence micrographs of dextranspermine compared to common transfection reagents in HEK293 and NIH3T3 cells. Adapted with permission from [38]. © 2002 American Chemical Society

Fig. 2

Structure of schizophyllan repeat unit and a schematic representation of the triple helix formed in aqueous solution. Figure adapted with permission from [45]. © 2003 Elsevier

Fig. 3

Structure of hyaluronan

Fig. 4

SEM images of DNA-HA matrices: (a) before and (b) after incubation in hyaluronidase solution (10 units/ml) for 7 days. Figure adapted with permission from [65]. © 2003 Elsevier

Fig. 5

Structure of pullulan

Fig. 6

Structure of DEAE-pullulan

Fig. 7

(a) Structure of chitosans. (b) Structure of chitosan-graft-PEI

Fig. 8

Synthesis of chitosan-graft-PEI. Figure reproduced with permission from [109]. © 2007 Elsevier

Fig. 9

Structure of urocanic acid-modified chitosan

Fig. 10

Synthesis of thiolated chitosan. Figure reproduced with permission from [127]. © 2004 Elsevier

Fig. 11

(a) Structure of galactosylated chitosan. Figure reproduced with permission from [102]. © 1996 Elsevier. (b) Structure of lactose conjugated chitosan. Figure reproduced with permission from [134]. © 2006 American Chemical Society

Fig. 12

Generalized block diagram of PGAA design structure and structures of the 16-polymer library of PGAAs. These polymers allow the direct comparison of changes in amine stoichiometry, as well as hydroxyl number and stereochemistry, on biological properties. Figure adapted with permission from [186]. © 2006 John Wiley & Sons, Inc

Fig. 13

Transgene expression efficiency of PGAA polymers in multiple mammalian cell types. (a) G, D, and M polyplexes shown high levels of transfection in multiple cell types. PGAAs with four secondary amines/repeat unit in H9c2 cells in (b) serum-free and (c) serum-containing media. Figures adapted with permissions from [21] and [146]. © 2005 and 2006 American Chemical Society

Fig. 14

Structures of linear PGAAs

Fig. 15

Internalization and gene expression of pDNA released from a multilayer assembly. Release of pDNA occurs upon degradation of T4 polyamide. Notable increase in fluorescence intensity over time is observed in the flow histograms. Gene expression (measured by intracellular GFP fluorescence) does not increase at the same rate as DNA uptake. Figure adapted with permission from [151]. © 2009 Elsevier

Fig. 16

Structures of trehalose-containing copolymers. Figure adapted with permission from [138]. © 2003 American Chemical Society

Fig. 17

Structures of trehalose click polymers

Fig. 18

Circular dichroism spectra comparing (a) Tr1 and (b) Tr4. Titration of pDNA with Tr1 results in minimal change in molar ellipticity representative of B-form DNA. Tr4 elicits a shift in ellipticity to a modified B-form, suggesting interaction with DNA base pairs by the polymer. Figure adapted with permission from [156]. © 2008 American Chemical Society

Fig. 19

Cellular internalization, transgene expression, and relative cell viability of Tr4. Cellular uptake in (a) serum-free and (b) serum-containing media. Transgene expression and cell viability in (c) HeLa and (d) H9c2(2-1) cells. Figure adapted with permission from [155]. © 2007 American Chemical Society

Fig. 20

Synthesis of β-CD-based polymer. Reproduced with permission from [141]. © 2001 American Chemical Society

Fig. 21

Structure of β-CD “click” polymers. Figure reproduced with permission from [173]. © 2009 American Chemical Society

Fig. 22

(a) Structure of β-cyclodextrin-containing polycations (CDP)s and (b) formulation of siRNA-containing targeted nanoparticles formed with CDP. Figure reprinted with permission from [177]. © 2007 National Academy of Sciences, U.S.A

Fig. 23

In vivo performance of targeted cyclodextrin polycations. (a) Growth curves for engrafted tumors. The median integrated tumor bioluminescent signal (photons/s) for each treatment group (n = 8–10) is plotted vs time after cell injection (days). (b) MRI confirmation of tumor engraftment. (c) Dose-dependent effects on cytokine production in non-human primates. Only very high dosage led to significant increases in cytokines. (ab) reprinted with permission from [174], figures 3 and 5. © 2005 American Association for Cancer Research. (c) reprinted with permission from [177]. © 2007 National Academy of Sciences, U.S.A

Fig. 24

Structure of PEGylated glycopeptide. Figure reproduced with permission from [182]. © 2007 American Chemical Society

Fig. 25

Characterization and luciferase expression of PGP DNA condensates in vivo. These results show that luciferase expression is dependent on galactose incorporation but independent of amount of melittin. (a) Represents the input mol ratio of Cys-terminated melittin, PEG-peptide, and glycopeptide. (b) Represents the measured mol ratio of Cys-terminated melittin, PEG-peptide, and glycopeptide for each purified PGP. (c) Values are the calculated MW based on polylysine standards. (d) Values are the calculated MW based on PEG standards. (e) The mean particle size determined at a stoichiometry of 0.3 nmol of PGP per mg of DNA. The value represents the mean diameter (nm) based on unimodal analysis. (f) The zeta potential of PGP DNA condensates at a stoichiometry of 0.3 nmol of PGP per mg of DNA. (g) The metabolic half-life of PGP 125I-DNA in triplicate mice. The results are derived from Fig. 6. (h) The PC/NPC ratio of DNA-targeted liver. (i) Represents a control PGP 3 in which galactose has been removed. Figure adapted with permission from [182]. © 2007 American Chemical Society

Fig. 26

Specific delivery of siRNA to hepatocytes with Dynamic PolyConjugates. (a) Confocal micrographs indicate specific intracellular delivery of oligonucleotides by targeting hepatocytes with N-acetylgalactosamine, as Cy3-labeled oligonucleotide (red) is seen within mouse hepatocytes, compared to when mannose and glucose are used as targeting moieties and Cy3 oligonucleotides are seen in the pericellular regions. (b) RT-qPCR shows dose-dependent decrease in apoB mRNA, corresponding to (c) decreasing serum cholesterol levels. (d) Increased hepatic lipid content (stained with oil red) relative to control siRNA and saline injections confirm knockdown of apoB-mediated cholesterol transport from the liver. Figure adapted with permission from [183]. © 2007 National Academy of Sciences, U.S.A