Gene Therapy (2014) 21, 212–218 & 2014 Macmillan Publishers Limited All rights reserved 0969-7128/14 www.nature.com/gt
ORIGINAL ARTICLE
Design of magnetic polyplexes taken up efficiently by dendritic cell for enhanced DNA vaccine delivery FM Nawwab AL-Deen1, C Selomulya1, YY Kong2, SD Xiang2, C Ma3, RL Coppel3,4 and M Plebanski2,4 Dendritic cells (DC) targeting vaccines require high efficiency for uptake, followed by DC activation and maturation. We used magnetic vectors comprising polyethylenimine (PEI)-coated superparamagnetic iron oxide nanoparticles, with hyaluronic acid (HA) of different molecular weights (o10 and 900 kDa) to reduce cytotoxicity and to facilitate endocytosis of particles into DCs via specific surface receptors. DNA encoding Plasmodium yoelii merozoite surface protein 1–19 and a plasmid encoding yellow fluorescent gene were added to the magnetic complexes with various % charge ratios of HA: PEI. The presence of magnetic fields significantly enhanced DC transfection and maturation. Vectors containing a high-molecular-weight HA with 100% charge ratio of HA: PEI yielded a better transfection efficiency than others. This phenomenon was attributed to their longer molecular chains and higher mucoadhesive properties aiding DNA condensation and stability. Insights gained should improve the design of more effective DNA vaccine delivery systems. Gene Therapy (2014) 21, 212–218; doi:10.1038/gt.2013.77; published online 19 December 2013 Keywords: dendritic cells; SPIONs; hyaluronic acid; DNA vaccine delivery
INTRODUCTION A valuable characteristic of DNA vaccination is the capacity to induce both cellular and antibody immunity.1 This is particularly useful when attempting to protect against diseases where an effective cellular immune response is crucial in controlling intracellular pathogens, including malaria, AIDS and tuberculosis.2 Various nonviral vectors have been utilized to deliver genes in vitro and in vivo,3 including lipid-based and polymercoated gene vectors. High levels of gene expression in vitro were generated with biodegradable poly(lactic-co-glycolic acid) microparticles of polyethylenimine (PEI)-condensed plasmid DNA encoding the blood-stage malaria protein PyMSP4/5,4 attributed to the narrow particle size distribution (0.8–1.9 mm) and high DNA encapsulation efficiencies (82–96%). The same group subsequently reported high levels of gene expression and moderate cytotoxicity in COS-7 cells using a similar carrier with plasmid DNA encoding PyMSP1–19 formulated via ultrasonic atomization.5 Previously, we have shown that magnetofection significantly improved the transfection efficiency of DNA encoding P. yoelii merozoite surface protein1–19 (MSP1–19) (VR1020-PyMSP1–19) in COS-7 cells.6 The vector compositions of superparamagnetic iron oxide nanoparticles (SPIONs) and PEI complexed under acidic condition showed better DNA transfection than at neutral condition, possibly due to the protonated structures of PEI polymer that entrapped and condensed higher amounts of DNA. More importantly, the application of an external magnetic field during gene transfection process dramatically increased the COS-7 cell transfection efficiency.6 It is to be noted that the transfection of COS cells can only be indicative of the potential of any DNA particle-based vaccine. However, to transfect specialized
antigen presenting cells (APCs) involved in inducing immunity, and specifically the critical primers of immunity, the dendritic cells (DCs), which contain unique particle uptake pathways, many of which are not present in COS cells.7 Insights into optimizing the transfection of DC, the most powerful APC, are needed if we are to develop effective vaccines that target DC in vivo as well as to optimize DC transfection for use in ex vivo therapies. Although the uptake of DNA or DNA-loaded vaccine carriers by DCs is a vital step for the transfection of these cells, it is by itself not enough to ensure a successful expression of the antigen in DC. More importantly, it should be also ensured that the DC cells are activated and able to interact positively with T cells, usually monitored by surface expression of CD86 and major histocompatibility complex (MHC) class II molecules. In general, the transfection efficiency of nonviral gene delivery systems in non-dividing primary cells like DCs decreases considerably when compared with transformed cell lines.8 A study recently done by Chapman et al.9 found that the application of permanent and pulsating magnetic fields could significantly enhance the efficiency of gene delivery in primary cells such as macrophages, synoviocytes, chondrocytes and others isolated from different organs if PEI-coated magnetic nanoparticles were used. To achieve optimal gene transfection specifically for DCs through improved cell entry, various barriers including DCs cellular targeting, particle uptake and DNA endosomal escape have to be overcome. DCs further need to be in a mature/activated state to express high levels of major histocompatibility molecules MHCI and MHCII, as well as T-cell costimulatory molecules such as CD86, to effectively stimulate CD8 and CD4 T-cell immunity. With respect to transfection of DCs, certain
1 Department of Chemical Engineering, Monash University, Clayton, Victoria, Australia; 2Department of Immunology, Central Clinical School, Faculty of Medicine, Nursing and Health Sciences, Monash University, Melbourne, Victoria, Australia and 3Department of Microbiology, Monash University, Clayton, Victoria, Australia. Correspondence: Dr C Selomulya, Department of Chemical Engineering, Monash University, Clayton Campus, Wellington Road, Clayton, Victoria 3800, Australia. E-mail: [email protected] 4 These authors contributed equally to this work. Received 16 April 2013; revised 15 October 2013; accepted 18 November 2013; published online 19 December 2013
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213 of targeting by combining magnetic nanoparticles, which initiate the surface of DCs under an external magnetic field, with targeting of specific DC receptors by HA ligands on the surface of the magnetic vectors. Here for the first time we report the results of utilizing the system of SPIONs/PEI/DNA-HA malaria gene complexes to target and transfect DC. Given the magnetic core, and the potential opportunity offered for directional transfection, we have further analyzed transfection efficacy with or without the added variable of an external magnetic field. In addition, we assessed the maturation/activation state of the DCs in vitro after interaction with these nanoparticles and magnetic fields. We focused on a quaternary system for blood-stage malaria DNA vaccine that encodes P. yoelii merozoite surface protein MSP1–19 (VR1020PyMSP1–19). The effects of combining different molecular weights of HA with different percentage charge ratios of HA: PEI on stability, cytotoxicity and DC transfection/maturation efficiency of the polyplexes were all investigated in vitro. This work illustrates how interactions of such well-designed quaternary complexes
plasmid DNA formulations or delivery systems may be able to target DCs for improved DNA uptake—for example, polyplexes with longer diblock copolymer chains showed the highest cellular uptake by DCs compared with short chain followed by effective gene transfection.10 Hyaluronic acid (HA) is a high-molecular-weight (HMW) mucopolysaccharide naturally present in all living organisms.11 It consists of a linear, unbranched, polyanions formed by D-glucuronic acid and N-acetyl-D-glucosamine repetitive units, proven to be low in toxicity, non-antigenic, non-immunogenic, biodegradable and biocompatible.11 Toll-like receptors (TLR) including TLR4 and TLR2, CD44, RHAMM, TNFIP6, HARE and LYVE-1 have been described as the main receptors for HA for different biological functions.12 A gene delivery system consisting of SPIONs/PEI/DNA-HA polyplexes can therefore be used to target DCs. The uptake of these polyplexes would be facilitated by HA receptor-mediated endocytosis on the surface of DCs, while at the same time maintaining the endosomal escape capacity of the PEI polymer.13 Such a system could provide an additional layer
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Figure 1. YFP expression and maturation of CD11c þ DCs induced by addition of functionalized DNA complexes to cells at a mass of 2 mg per well. DCs were isolated from the old C57BL/6 mice and incubated with different DNA complexes. After 24 h, cells were harvested and stained for expressions of CD86, MHC class I and MHC class II and analyzed by flow cytometry: (a) representative set of flow cytometry histograms of CD86 expression on CD11c þ DCs expressing YFP. Gray shaded area: untreated cells; (b) CD86 maturation markers on CD11c þ DCs expressing YFP; (c) YFP expression level and MHC I, MHC II costimulatory molecule expression on CD11c þ DCs. Data are analyzed statistically using one-way analysis of variance (ANOVA), and the comparison of means conducted using Tukey multiple comparison test. Results are expressed as means±s.d. (n ¼ 3). *Po0.05, **Po0.001, ***Po0.0001 (w magnet indicates application of an external magnetic field; w/o magnet indicates without application of an external magnetic field). & 2014 Macmillan Publishers Limited
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214 under magnetic field may markedly influence the delivery of genetic cargos to DCs. RESULTS The detailed characterization data of SPIONs/PEI/DNA-HA polyplexes as well as cell viability as measured by MTT (3-[4,5dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assays in COS-7 cells treated with these polyplexes can be found in Supplementary Information (Supplementary Figures S1–S6). The rationale of selecting this system has been discussed in our recent work.14 We examined the effects of magnetofection using quaternary polyplexes on mouse bone marrow-derived DC transfection and subsequent maturation (assessed by expression of MHCI, MHCII and CD86) after 5 h incubation with 2 h application of an external magnetic field. As shown in Supplementary Figure S6, the typical purity of CD11c þ DCs, which is the transmembrane protein found at high levels on most DCs, was B70–80%. Treatments with different DNA complexes, especially quaternary polyplexes, show no significant negative effects on the expression of CD11c on DCs compared with the normal control. Internalization of exogenous antigens is a prerequisite for APCs transfection with the antigen of interest and would improve their antigen presentation to T cells. Magnetofection-based DCs transfection and maturation in vitro Flow cytometry analysis results of yellow fluorescent protein (YFP) expression in CD11c þ DCs show that almost all SPIONs/PEI/ DNA-HA polyplexes increased significantly the transfection efficiency under the magnetic field, compared with transfection with other DNA complexes and even quaternary complexes without an applied magnetic field, regardless of the HA: PEI charge ratio or HA molecular weight (Figure 1). A trend was further observed between DC cellular uptake via magnetofection and increasing the molecular weight of HA in the quaternary polyplexes. Effect of HA blocking on cellular uptake of polyplexes with HA receptors Flow cytometric analysis results of YFP expression in CD11c þ DCs show that the DC pretreated with excess HA ligand has nearly 50% reduction in YFP gene expression compared with the transfection with cells pretreated with phosphate-buffered saline (PBS) alone (Figure 2a). Figure 2b shows that all wells treated with quaternary
HA-coated polyplexes induced activation of DC independently of CD44 We assessed the CD44 expresssion level on total CD11c þ DCs and CD11c þ DCs expressing YFP after treatment with different gene complexes compared with CD44 expression level in untreated CD11c þ DCs. Our results show strong expression of surface CD44 receptor around 90% in all CD11c þ DCs indicating the presence of this type of receptor in our DC cultures (Figure 3).
DISCUSSION DCs transfection and maturation An important concern resulting in insufficient gene delivery of nonviral gene vectors in vitro is the low accumulation of gene complexes at the cell surface. Here we applied an external magnetic field on the quaternary magnetic gene vector to accumulate the gene vector on DCs in the culture media.15,16 We used DNA encoding a fluorescent protein (YFP) mixed in 1:1 ratio with the MSP1–19 expressing vector to allow us to read out the efficiency of DNA uptake. Western blot analysis using antiPyMSP1–19 antibodies was further performed on transfected DCs pellet to identify the identity of recombinant proteins produced by PyMSP1–19 plasmid in CD11c þ DCs. The result shows that the expressed protein in DCs is equal to the PyMSP1–19 protein expressed in Escherichia coli (Supplementary Figure S7). The ability of DCs to drive antigen-specific immunity is dependent on their degree of maturation. Flow cytometric analyses provided evidence that generally, the treatment of DCs with plasmid DNA or even plasmid DNA loaded onto nanoparticles in general enhanced the expression of CD86 in CD11c þ DCs expressing YFP. This is because bacterial plasmid DNA possesses inherent immune adjuvant activity based on its unmethylated CpG motifs in the plasmid DNA backbone, previously shown to act as a potent DNA vaccine adjuvant in rodents.17 Culturing DCs with quaternary polyplexes induced substantial upregulation of CD86 in the overall culture, it specifically increased both efficiency of YFP gene transfection in CD11c þ DCs and concomitant maturation through upregulated expression of costimulatory molecule CD86 and MHC molecules (Figures 1b and c).
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polyplexes pretreated with PBS alone (without HA) upregulated CD86 maturation marker on CD11c þ DCs expressing YFP regardless of the HA molecular weight, in contrast to pretreated HA wells.
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Figure 2. Effects of HA-pre-addition on the YFP expression and costimulatory surface marker (CD86) on CD11c þ DCs mediated by quaternary polyplexes: (a) the percentage of total CD11c þ DC cells expressing YFP; (b) the percentage of CD11c þ DCs expressing YFP with upregulation of CD86 maturation signal (n ¼ 3). ***Po0.0001 (w magnet indicates application of an external magnetic field; w/o magnet indicates without application of an external magnetic field). Gene Therapy (2014) 212 – 218
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Figure 3. Effects of different DNA complexes on the expression of CD44 receptors on CD11c þ DCs population: (a) the percentage of total CD11c þ DC cells; (b) the percentage of CD11c þ DCs expressing YFP. Data are analyzed statistically using one-way ANOVA, and the comparison of means was conducted using the Tukey multiple comparison test. Results are expressed as means ±s.d. (n ¼ 3). ***Po0.001 (w magnet indicates application of an external magnetic field; w/o magnet indicates without application of an external magnetic field).
In contrast, DCs treatment with different DNA complexes excluding quaternary polyplexes did not significantly enhance YFP gene expression and also the level of stimulatory markers (CD86, MHC molecules) on the CD11c þ DCs expressing YFP, suggesting that internalization is the main contributor in quaternary polyplexes-induced concomitant DC gene transfection and maturation (Figures 1a and b). The YFP gene expression and phenotypical maturation signals observed during DCs exposure to quaternary polyplexes were much higher compared with those of ternary polyplexes. These results support the notion that factors such as endocytosis via HA receptors may have a role in DNA complexes-induced DC maturation, as HA was present in the quaternary system. In addition, unmethylated CpG motifs of plasmid DNA are known to be recognized by the TLR9 in DCs which have an important role in long-term vaccination protocols.18 It is likely that upon endocytosis, plasmid DNA becomes at least partially degraded by DNase II in the endosomal-lysosomal compartment to release CpG motifs. As TLR9 is not present on the surface of the cells, targeted delivery of plasmid DNA by means of nanoparticles to the endosomal compartment might be important for potential association of DNA with the TLR9 receptor in the endosome and trigger DC maturation followed by the immune response. The effects of polyplexes gene transfection on DCs transfection and maturation in the in vitro culture correlated with the extent of in vivo DC maturation through the consequent adjuvant effect on the immune response toward the antigen of interest. The data obtained highlighted (for the first time) to the direct interactions of DC with magnetic gene complexes coated by PEI and HA polymers in promoting DCs maturation. Therefore, suitable surface modifications by HA should allow specific endocytosis of gene complexes, to affect the pathways of intracellular processing and trafficking. It also suggests that the prime mechanism by which the gene complexes enhance an adaptive immune response is maturation of the APCs, specifically DC, such that they become efficient at antigen presentation and T-cell stimulation to generate an immune response to the antigen of interest. As these results suggest that quaternary polyplexes were stimuli of DC maturation through internalization, probably by specific HA receptor, we sought to further investigate the effects of HA molecular weight and magnetic field applications on the uptake of quaternary polyplexes by murine DCs, to determine to what extent the particle endocytosis had a role in DC maturation. As shown in Figures 1a and b, SPIONs/PEI/DNA-HA polyplexes at HMW HA and 100% charge ratio of HA: PEI (100% HMW polyplexes) under magnetic field display the highest YFP gene & 2014 Macmillan Publishers Limited
expression, accompanied also by maturation through upregulated expression of CD86, compared with transfection with other complexes. The efficiency is much higher than DNA alone (Po0.0001). Although SPIONs/PEI/DNA ternary polyplexesmediated transfection was also done under a magnetic field, they showed significantly lower transfection efficiency than SPIONs/ PEI/DNA-HA quaternary polyplexes and DNA-Lipofectamine 2000 (a standard gene delivery agent) (Po0.05). Interestingly, 100% HMW polyplexes show approximately the same levels of YFP and CD86 expression under or in the absence of magnetic field. The physicochemical properties of HA are related to its molecular weight.19 Higher molecular weight is associated with longer molecular chains that in turn increase the mucoadhesive properties of HA in solution. Lim et al.20 also found that the incorporation of HA with molecular weight of 8.5 105 Da into macro-particles increased mucoadhesive properties more than chitosan-based systems. HMW HA in quaternary polyplexes is likely to increase the mucoadhesive and viscoelastic properties of the complexes, accordingly enhances large molecular chains of HA ligands accessibility to the DC receptors and promotes the multivalent binding to receptors,21 regardless of the presence or absence of the magnetic field. The efficiency of HA polymer with HMW in accessing DC cells is associated with their ability to bind to DCs receptors, with a previous study showing that HMW HA binding was irreversible to CD44 cell receptors, although the binding became weaker and reversible with decreased HA molecular weight.22 On the other hand, applying an external magnetic field could enhance the accumulation and attachment of low molecular weight (LMW) quaternary polyplexes on the surface of DCs, resulting in significant increases of the cellular uptake of the complex (Po0.001), accompanied by improved phenotypic maturation of DCs.23 Indeed, the effects of magnetic field on LMW (LMW HA) quaternary polyplexes are more pronounced in the 5% LMW polyplexes (Figure 1b). This new observation is in good agreement with a previous study by Qhattal et al.24 showing that DC cellular targeting efficiency of HA-liposomes nanoparticles depended strongly upon HA molecular weight and grafting density, with HMW HA resulting in better cellular adhesion and internalization of HA-liposomes to DCs. In this study, the concentration of HA is in the range of 0.012–0.14 mg ml 1, approximately similar to the range used by Do et al.25 who found that the addition of 0.01–1 mg ml 1 of HA in DCs culture were very effective in growing cells, whereas high HA concentration of 0.5 mg ml 1 inhibited the growth of DCs. In agreement with our data, the polyplexes with these Gene Therapy (2014) 212 – 218
Dendritic cells-targeting DNA vaccine FN Al-Deen et al
216 concentrations could interact properly with DCs and promote their maturation. In addition, our goal was to determine whether HA-coated quaternary polyplexes could increase the functional uptake of DNA by DCs for targeted gene expression and antigen presentation. After DC transfection, the produced antigen was processed and presented by MHC class I and MHC class II to stimulate cytotoxic T-cells and helper T cells, respectively.26 These intracellular produced antigens by DCs are hard to detect and present as MHC class I and II, as the molecular endocytosis of antigen is not necessarily sufficient to ensure efficient antigen presentation.27 However, we showed that the quaternary polyplexes considerably enhanced antigen uptake and presentation by DCs, probably via HA-mediated endocytosis that has been enhanced by the application of external magnetic field in vitro (as shown in Figure 1c) and via initial in vivo studies (Supplementary Figure S8). The use of quaternary polyplexes under magnetic field significantly upregulated MHC class I and MHC class II expressions on DCs, supporting further their advanced maturation profile (Figure 1c). HA molecules may also act as a condensation agent for DNA in a similar manner to PEI, especially as DNA molecules were mixed with HA polymer before addition to SPION-PEI complexes. Kim et al.28 showed by gel electrophoresis that DNA could interact to some degree with HA fragments, with only small portions of the DNA molecules able to be released from the DNA-HA matrix. It is also known that the positively charged PEI condenses DNA into a compact colloid while affording a good protection from DNase digestion.13 Thus, the interaction of DNA with HA and then condensation together with PEI-coated SPIONs could well increase the stability of DNA in the complexes, and at the same time protect DNA from DNase degradation. However, strong interactions between PEI and DNA may delay DNA release from complexes or even prevent all DNA reaching the nucleus, leading to a decrease in transfection efficiency and gene expression. HA might be useful in this regard as well, behaving here like a transcriptional activator by lessening the electrostatic complexation between DNA and PEI polymer (refer to Supplementary Information and Supplementary Figure S4A, gel retardation assay), thus facilitating the interaction of transcription factors with plasmid DNA. Granulocyte macrophage colony stimulating factor (GM-CSF) was added at a final concentration of 10 ng ml 1 to the cell culture in order to stimulate DC differentiation. Adding this cytokine to DC culture has been described previously to activate DCs proliferation and cellular uptake.29 Adding this cytokine to the cell culture with the quaternary polyplexes might promote cellular uptake of the gene complexes, followed by gene expression in the DCs. Our study result seemed to confirm the finding by Ali et al.30 in suggesting that placing DC in the media containing low levels of GM-CSF (50 ng ml 1) during PEI-DNA gene transfection could greatly enhance gene transfection, comparatively to high amounts of GM-CSF (500 ng ml 1) that could promote the degradation of intercellular DNA and its protein products. Modifying the magnetic gene vector with HA polymer also led to higher stability of complexes in the DCs media. This could lead to more cellular uptake of the complexes with well-timed dissociation of the DNA–polymer complex. Other possible mechanisms of high gene expression relate to the presence of HA, was the notably decreased cytotoxicity and the size of quaternary polyplexes in the cell media to around 150 nm at the optimal charge ratio of 100%, leading to better distribution with high cellular uptake.31 Effect of HA blocking on cellular uptake The YFP gene expression in CD11c þ DCs treated with HMW polyplexes without pretreated HA showed significantly the highest gene expression (Po0.0001), whereas LMW HA polyplexes Gene Therapy (2014) 212 – 218
with and without pretreated HA did not show any significant difference in gene expression (Figure 2a). This suggested that the YFP expression of HMW polyplexes could be mediated by HMW HA-specific receptors on the surface of DC cells, more than the LMW HA that could be competitively inhibited by excess free HA. The trend shown in Figure 2b is in agreement with Ito et al.,32 who showed that the CHO cells pretreated by HA had diminished gene expression compared with highly enhanced luciferase gene expression of HA-coated ternary complexes (DNA/PE/HA) pretreated with only PBS. Taken together, these results suggested interactions between HA special receptors on DC specifically with HMW HA-coated polyplexes that could encourage uptake via these receptors.32 Activation of DC independently of CD44 CD44 is a principal cellular HA receptor,12,33 raising the question on whether CD44 could also mediates the HA-coated quaternary polyplexes uptake. In general, untreated or treated CD11c þ DCs with different DNA polyplexes showed approximately the same percentage of CD44 expression. The ternary polyplexes, DNA/Lipofectamine complexes and even free DNA slightly, but not significantly, upregulated CD44 expression level on DCs especially with cells expressed YFP compared with control DCs. In contrast, DC pretreated with high concentration of HA before adding the quaternary polyplexes showed significant trends toward decreasing CD44 in total DCs or DCs expressed YFP compared with control DCs (Figures 3a and b). Adding HA at high concentration may potentially inhibit CD44 expression, as previous study of Kaya et al.34 suggested that the abnormal accumulation of hyaluronate in superficial dermis due to some medical reasons was accompanied by the decline in CD44 expression in the keratinocytes, and it could be argued that a similar mechanism could promote low CD44 expression on DCs when pretreated with high amounts of HA (Figure 3). A recent report showed that under normal condition, CD44 receptors are rapidly recycled back from the intracellular compartment to the plasma membrane following internalization and release of cargos.35 Therefore, the possibility of a lower expression of CD44 due to uptake of the HA-coated quaternary polyplexes as a receptor for HA could be excluded, as the fluorescence-activated cell sorting analysis was performed 24 h after adding the polyplexes in the cell culture, allowing enough time for CD44 to recycle back to the cell surface. The transfection study (Figure 1) shows that DCs treatment with the quaternary polyplexes especially under magnetic field displays significantly high levels of YFP gene expression accompanied by significant upregulation in the CD86 expression; thus, implying that quaternary polyplexes were actively uptaken by DCs via different type of HA receptors than CD44. Most interestingly, there is no significant difference in CD44 expression levels between total CD11c þ DCs and CD11c þ DCs transfected with YFP gene for cells treated with different HA-coated quaternary polyplexes compared with control DCs. Taken together, these data potentially demonstrate that CD44 receptors were not actively involved in HA quaternary polyplex uptake by DCs, thereby suggesting a possible role for other receptors of HA, such as TLR2 and TLR4, CD38, LYVE-1 and RHAMM or even unknown novel receptors.12 Others have suggested that the mammalian endocytic HA receptor is HARE instead of CD44.36 Furthermore, Do et al.25 previously reported that HA could activate DC through CD44-independent pathway, although CD44 on antigen-specific T-cells had a critical role during interaction with DC to present antigen and subsequently T-cells expansion. Moreover, Termeer et al.37 found that CD44KO-derived DCs (knockout mice) showed similar upregulation of costimulatory molecules like wild-type-derived DCs after HA treatment. It should be noted that these studies were different from the current study in that they primarily investigated the ability of HA-treated DCs to & 2014 Macmillan Publishers Limited
Dendritic cells-targeting DNA vaccine FN Al-Deen et al
217 activate alloreactive T-cells. Our aim here was to transfect DCs using malaria DNA vaccine condensed on HA-coated magnetic quaternary polyplexes and at the same time to increase DCs maturation through upregulation of costimulatory molecules. HA on the surface of these quaternary complexes could act as ligands to enhance receptor-mediated endocytosis through specific HA receptors on DCs. However, we cannot eliminate the possibility that CD44 could make a minor contribution to the cellular uptake of quaternary polyplexes involved in the polyplexes transfection and maturation of wild-type-derived DCs. As we did not detect expressions by other receptors in mature or immature DC by reverse transcriptase-PCR, we could exclude the effects of other HA receptors. Further study will be necessary to determine the specific type of DCs receptor contributing to the uptake of quaternary polyplexes. Although it is reported in the literature that DC is problematic to transfect, here we demonstrate that quaternary magnetic polyplexes containing HA could be used to transfect DCs in vitro highly effectively with a model DNA-based malaria vaccine candidate. The transfection could be enhanced by increasing the HA molecular weight and the application of an external magnetic field. The size of HA ligand on the polyplexes was an important determinant of its cellular uptake, with higher molecular weight of HA resulting in better cellular adhesion and internalization. Moreover, the presence of HA loosened the strong complexation between DNA and PEI polymer, but did not result in DNA disassembly, with higher cell viability compared with those treated without HA in the polyplexes. These quaternary polyplexes also had adjuvant activity that encouraged DC maturation (specifically for the cells that had internalized the complexes), while the application of an external magnetic field improved gene delivery to DCs at low DNA doses, while also upregulating DC maturation markers. This new information will be crucial for the design of DNA gene delivery tool to somatic cells such as DCs for potent immune activation, with further studies currently underway to investigate the detailed uptake mechanisms and also the specific type of DCs receptor of this gene delivery system both in vitro and in vivo.
Supplementary Information. Bone marrow-derived DCs were generated as previously described with minor alterations.38
Magnetofection-based DC transfection and maturation in vitro To examine the effects of quaternary polyplexes on DCs transfection and maturation state, on day 5 of the bone marrow-derived DCs culture, culture plates were centrifuged and the medium was removed. The cells in each well were washed with serum-free RPMI 1640 medium. Freshly prepared ternary and quaternary polyplexes of SPIONs/PEI/DNA-HA with two different molecular weights of HA and two different ratios of HA: PEI (charge) were diluted in serum-free RPMI 1640 medium for 15 min at room temperature. The polyplexes with N/P of 10 were then added to the cells with a fixed amount of MSP1–19/YFP plasmid mixtures of 2 mg per well. The cells were cultured with a neodymium-iron-boron magnet positioned under each well for 2 h. After 5 h, the culture media containing polyplexes were removed from each well and replaced with 2 ml of the fresh complete medium containing 10 ng ml 1 (GM-CSF) and cultured for a further 24 h in 37 1C incubator with 5% CO2. DCs transfection efficiency by quaternary polyplexes was examined with or without the application of magnetic field during the process. Transfection of DCs with plasmid DNA alone without complexation in the polyplexes was also examined for comparison. To examine the transfection efficiency of quaternary polyplexes in DCs and maturation of CD11c þ DCs, the non-adherent cells were harvested and washed with PBS after 24 h. The cells were stained using the following antibodies: APC-CD11c, biotinylated-CD86, APC-CD44, PE-MHC Class I, APC-Cy7-MHC Class II, whereas dead cells were excluded using live/dead fixable dead cell stain kit (Aqua LIVE/DEAD, Invitrogen). Biotin-labeled monoclonal antibodies were developed with PE-Cy7-streptavidin and analyzed by flow cytometry (FACSCalibur). Untreated DCs were also included as negative controls. Quaternary polyplexes coated with HA can be easily dispersed in cell medium, favorably responding to receptor-ligand interactions with HA receptors positive-cells such as DCs, although they could also loosen strong electrostatic interactions between DNA and PEI (Supplementary Figure S4A). A way to prove the existence of these interactions was by deliberately blocking HA receptors on the cells by pretreatment with a saturable amount of free HA prior incubation with different plasmid complexes. Before adding different DNA polyplexes to the cells, 200 ml of HA solutions (3 mg ml 1 in PBS) (HMW or LMW) were added to DC cells in addition to 1 ml of RPMI medium. After incubation for 60 min at 37 1C, the cells were washed with RPMI media to remove unbound HA and then different complexes were added to the wells by application of external magnetic field for 2 h, with similar treatment as previously referred to in the transfection assay.
MATERIALS AND METHODS Materials Sodium hyaluronate (HA) with average molecular weight of o10 and 900 kDa were purchased from Life Core Biomedical LLC (Chaska, MN, USA). RPMI 1640 medium, 0.05% trypsin-EDTA, penicillin/streptomycin, L-glutamine, Lipofectamine and fetal calf serum 2000 were from GibcoBRL (Gathersburg, MD, USA). Hamster-anti-mouse APC-CD11c antibody, rat-anti-mouse biotinylated-CD86 (B7.2), rat-anti-mouse APC-CD44 and ratanti-mouse PE-MHC Class I were purchased from BD Pharmingen (Franklin Lakes, NJ, USA), whereas GM-CSF were ordered from PeproTech (Rocky Hill, NJ, USA). PE-Cy7-streptavidin was from BD Bioscience (Franklin Lakes, NJ, USA), whereas rat-anti-Mouse APC-Cy7-MHC Class II was from eBioscience (San Diego, CA, USA). Live/dead fixable dead cell stain kit (Aqua LIVE/DEAD; Invitrogen (Carlsbad, CA, USA)) and Lipofectamine 2000 (Gibco-BRL) were supplied by Invitrogen. Mammalian expression vector VR1020 (Vical Inc., San Diego, CA, USA), VR1020-PyMSP1–19 and VR1020-YFP plasmids were obtained from collaborators at the Coppel lab (Department of Microbiology, Monash University, Clayton, Victoria, Australia). C57BL/6 mice (aged 6–8 weeks) were supplied by the animal facilities in the Alfred Medical Research and Education Precinct (AMREP). Arrays of permanent magnets of neodymium-iron-boron (Nd-Fe-B) in the format of a six-well plate were used for in vitro transfection experiments. The magnets were circular disk Nd-Fe-B magnets (diameter 25 mm, height 5 mm) glued onto the bottom of a six-well plate. Detailed procedures for preparation of SPIONs/PEI and SPIONs/PEI/ DNA-HA quaternary polyplexes, purification of plasmids DNA, characterization of these polyplexes via size and zeta potential measurements, DNA retardation and DNase I sensitivity assays and the determination of COS-7 cell variability by MTT colorimetric assay can be found in the & 2014 Macmillan Publishers Limited
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We acknowledge Dr Tim Williams from the Monash Centre of Electron Microscopy for assistance in TEM imaging.
REFERENCES 1 Liu MA. DNA vaccines: an historical perspective and view to the future. Immunol Rev 2011; 239: 62–84. 2 Seder RA, Hill AVS. Vaccines against intracellular infections requiring cellular immunity. Nature 2000; 406: 793–798. 3 Park TG, Jeong JH, Kim SW. Current status of polymeric gene delivery systems. Adv Drug Deliv Rev 2006; 58: 467–486. 4 Liu S, Danquah MK, Ho J, Ma C, Wang L, Coppel R et al. Preparation and characterization of poly(lactic-co-glycolic acid) microparticles containing DNA molecules encoding a malaria vaccine candidate. J Chem Technol Biotechnol 2009; 84: 782–788. 5 Liu S, Danquah MK, Forde GM, Ma C, Wang L, Coppel R. Microparticlemediated gene delivery for the enhanced expression of a 19-KDa fragment of merozoite surface protein 1 of Plasmodium falciparum. Biotechnol Prog 2010; 26: 257–262. 6 Al-Deen FN, Ho J, Selomulya C, Ma C, Coppel R. Superparamagnetic nanoparticles for effective delivery of malaria DNA vaccine. Langmuir 2011; 27: 3703–3712.
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218 7 Xiang SD, Scalzo-Inguanti K, Minigo G, Park A, Hardy CL, Plebanski M. Promising particle-based vaccines in cancer therapy. Expert Rev Vaccines 2008; 7: 1103–1119. 8 Hamm A, Krott N, Breibach I, Blindt R, Bosserhoff AK. Efficient transfection method for primary cells. Tissue Engineering 2002; 8: 235–245. 9 Chapman SWK, Hassa PO, Koch-Schneidemann S, von Rechenberg B, Hofmann-Amtenbrink M, Steitz B et al. Application of pulsed-magnetic field enhances non-viral gene delivery in primary cells from different origins. J Magn Magn Mater 2008; 320: 1517–1527. 10 Tang RP, Palumbo RN, Nagarajan L, Krogstad E, Wang C. Well-defined block copolymers for gene delivery to dendritic cells: Probing the effect of polycation chain-length. J Control Release 2010; 142: 229–237. 11 Necas J, Bartosikova L, Brauner P, Kolar J. Hyaluronic acid (hyaluronan): a review. Veterinarni Medicina 2008; 53: 397–411. 12 Oh EJ, Park K, Kim KS, Kim J, Yang JA, Kong JH et al. Target specific and long-acting delivery of protein, peptide, and nucleotide therapeutics using hyaluronic acid derivatives. J Control Release 2010; 141: 2–12. 13 Godbey WT, Wu KK, Mikos AG. Poly(ethylenimine) and its role in gene delivery. J Control Release 1999; 60: 149–160. 14 Al-Deen FN, Selomulya C, Williams T. On designing stable magnetic vectors as carriers for malaria DNA vaccine. Colloids Interfaces B: Biointerfaces 2012; 102: 492–503. 15 Plank C, Schillinger U, Scherer F, Bergemann C, Remy JS, Krotz F et al. The magnetofection method: Using magnetic force to enhance gene delivery. Biol Chem 2003; 384: 737–747. 16 Scherer F, Anton M, Schillinger U, Henkel J, Bergemann C, Kruger A et al. Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. Gene Ther 2002; 9: 102–109. 17 Hartmann G, Weiner GJ, Krieg AM. CpG DNA: A potent signal for growth, activation, and maturation of human dendritic cells. Proc Natl Acad Sci USA 1999; 96: 9305–9310. 18 Spies B, Hochrein H, Vabulas M, Huster K, Busch DH, Schmitz F et al. Vaccination with plasmid DNA activates dendritic cells via toll-like receptor 9 (TLR9) but functions in TLR9-deficient mice. J Immunol 2003; 171: 5908–5912. 19 Liao YH, Jones SA, Forbes B, Martin GP, Brown MB. Hyaluronan: pharmaceutical characterization and drug delivery. Drug Delivery 2005; 12: 327–342. 20 Lim ST, Forbes B, Berry DJ, Martin GP, Brown MB. In vivo evaluation of novel hyaluronan/chitosan microparticulate delivery systems for the nasal delivery of gentamicin in rabbits. Int J Pharm 2002; 231: 73–82. 21 Laurent TC, Fraser JRE, Pertoft H, Smedsrod B. Binding of hyaluronate and chondroitin sulfate to liver endothelial-cells. Biochem J 1986; 234: 653–658. 22 Wolny PM, Banerji S, Gounou C, Brisson AR, Day AJ, Jackson DG et al. Analysis of cd44-hyaluronan interactions in an artificial membrane system insights into the distinct binding properties of high and low molecular weight hyaluronan. J Biol Chem 2010; 285: 30170–30180.
23 Yao J, Fan Y, Du RH, Zhou JP, Lu Y, Wang W et al. Amphoteric hyaluronic acid derivative for targeting gene delivery. Biomaterials 2010; 31: 9357–9365. 24 Qhattal HSS, Liu XL. Characterization of CD44-mediated cancer cell uptake and intracellular distribution of hyaluronan-grafted liposomes. Mol Pharm 2011; 8: 1233–1246. 25 Do Y, Nagarkatti PS, Nagarkatti M. Role of CD44 and hyaluronic acid (HA) in activation of alloreactive and antigen-specific T cells by bone marrow-derived dendritic cells. J Immunother 2004; 27: 1–12. 26 Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998; 392: 245–252. 27 Watts C. Capture and processing of exogenous antigens for presentation on MHC molecules. Ann Rev Immunol 1997; 15: 821–850. 28 Kim A, Checkla DM, Dehazya P, Chen WL. Characterization of DNA-hyaluronan matrix for sustained gene transfer. J Contro Release 2003; 90: 81–95. 29 Hamilton JA, Anderson GP. GM-CSF biology. Growth Factors 2004; 22: 225–231. 30 Ali OA, Mooney DJ. Sustained GM-CSF and PEI condensed pDNA presentation increases the level and duration of gene expression in dendritic cells. J Control Release 2008; 132: 273–278. 31 Manolova V, Flace A, Bauer M, Schwarz K, Saudan P, Bachmann MF. Nanoparticles target distinct dendritic cell populations according to their size. Eur J Immunol 2008; 38: 1404–1413. 32 Ito T, Iida-Tanaka N, Niidome T, Kawano T, Kubo K, Yoshikawa K et al. Hyaluronic acid and its derivative as a multi-functional gene expression enhancer: protection from non-specific interactions, adhesion to targeted cells, and transcriptional activation. J Control Release 2006; 112: 382–388. 33 Culty M, Nguyen HA, Underhill CB. The hyaluronan receptor (cd44) participates in the uptake and degradation of hyaluronan. J Cell Biol 1992; 116: 1055–1062. 34 Kaya G, Augsburger E, Stamenkovic I, Saurat JH. Decrease in epidermal CD44 expression as a potential mechanism for abnormal hyaluronate accumulation in superficial dermis in lichen sclerosus et atrophicus. J Invest Dermatol 2000; 115: 1054–1058. 35 Garg HG, Hales CA. Chemistry and Biology of Hyaluronan. Elsevier Ltd.: Oxford, UK, 2004. 36 Weigel JA, Raymond RC, Weigel PH. The hyaluronan receptor for endocytosis (HARE) is not CD44 or CD54 (ICAM-1). Biochem Biophys Res Commun 2002; 294: 918–922. 37 Termeer CC, Hennies J, Voith U, Ahrens T, Weiss JM, Prehm P et al. Oligosaccharides of hyaluronan are potent activators of dendritic cells. J Immunol 2000; 165: 1863–1870. 38 Inaba K, Inaba M, Deguchi M, Hagi K, Yasumizu R, Ikehara S et al. Granulocytes, macrophages, and dendritic cells arise from a common major histocompatibility complex class-ii-negative progenitor in mouse bone-marrow. Proc Natl Acad Sci USA 1993; 90: 3038–3042.
Supplementary Information accompanies this paper on Gene Therapy website (http://www.nature.com/gt)
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ORIGINAL ARTICLE
Design of magnetic polyplexes taken up efficiently by dendritic cell for enhanced DNA vaccine delivery FM Nawwab AL-Deen1, C Selomulya1, YY Kong2, SD Xiang2, C Ma3, RL Coppel3,4 and M Plebanski2,4 Dendritic cells (DC) targeting vaccines require high efficiency for uptake, followed by DC activation and maturation. We used magnetic vectors comprising polyethylenimine (PEI)-coated superparamagnetic iron oxide nanoparticles, with hyaluronic acid (HA) of different molecular weights (o10 and 900 kDa) to reduce cytotoxicity and to facilitate endocytosis of particles into DCs via specific surface receptors. DNA encoding Plasmodium yoelii merozoite surface protein 1–19 and a plasmid encoding yellow fluorescent gene were added to the magnetic complexes with various % charge ratios of HA: PEI. The presence of magnetic fields significantly enhanced DC transfection and maturation. Vectors containing a high-molecular-weight HA with 100% charge ratio of HA: PEI yielded a better transfection efficiency than others. This phenomenon was attributed to their longer molecular chains and higher mucoadhesive properties aiding DNA condensation and stability. Insights gained should improve the design of more effective DNA vaccine delivery systems. Gene Therapy (2014) 21, 212–218; doi:10.1038/gt.2013.77; published online 19 December 2013 Keywords: dendritic cells; SPIONs; hyaluronic acid; DNA vaccine delivery
INTRODUCTION A valuable characteristic of DNA vaccination is the capacity to induce both cellular and antibody immunity.1 This is particularly useful when attempting to protect against diseases where an effective cellular immune response is crucial in controlling intracellular pathogens, including malaria, AIDS and tuberculosis.2 Various nonviral vectors have been utilized to deliver genes in vitro and in vivo,3 including lipid-based and polymercoated gene vectors. High levels of gene expression in vitro were generated with biodegradable poly(lactic-co-glycolic acid) microparticles of polyethylenimine (PEI)-condensed plasmid DNA encoding the blood-stage malaria protein PyMSP4/5,4 attributed to the narrow particle size distribution (0.8–1.9 mm) and high DNA encapsulation efficiencies (82–96%). The same group subsequently reported high levels of gene expression and moderate cytotoxicity in COS-7 cells using a similar carrier with plasmid DNA encoding PyMSP1–19 formulated via ultrasonic atomization.5 Previously, we have shown that magnetofection significantly improved the transfection efficiency of DNA encoding P. yoelii merozoite surface protein1–19 (MSP1–19) (VR1020-PyMSP1–19) in COS-7 cells.6 The vector compositions of superparamagnetic iron oxide nanoparticles (SPIONs) and PEI complexed under acidic condition showed better DNA transfection than at neutral condition, possibly due to the protonated structures of PEI polymer that entrapped and condensed higher amounts of DNA. More importantly, the application of an external magnetic field during gene transfection process dramatically increased the COS-7 cell transfection efficiency.6 It is to be noted that the transfection of COS cells can only be indicative of the potential of any DNA particle-based vaccine. However, to transfect specialized
antigen presenting cells (APCs) involved in inducing immunity, and specifically the critical primers of immunity, the dendritic cells (DCs), which contain unique particle uptake pathways, many of which are not present in COS cells.7 Insights into optimizing the transfection of DC, the most powerful APC, are needed if we are to develop effective vaccines that target DC in vivo as well as to optimize DC transfection for use in ex vivo therapies. Although the uptake of DNA or DNA-loaded vaccine carriers by DCs is a vital step for the transfection of these cells, it is by itself not enough to ensure a successful expression of the antigen in DC. More importantly, it should be also ensured that the DC cells are activated and able to interact positively with T cells, usually monitored by surface expression of CD86 and major histocompatibility complex (MHC) class II molecules. In general, the transfection efficiency of nonviral gene delivery systems in non-dividing primary cells like DCs decreases considerably when compared with transformed cell lines.8 A study recently done by Chapman et al.9 found that the application of permanent and pulsating magnetic fields could significantly enhance the efficiency of gene delivery in primary cells such as macrophages, synoviocytes, chondrocytes and others isolated from different organs if PEI-coated magnetic nanoparticles were used. To achieve optimal gene transfection specifically for DCs through improved cell entry, various barriers including DCs cellular targeting, particle uptake and DNA endosomal escape have to be overcome. DCs further need to be in a mature/activated state to express high levels of major histocompatibility molecules MHCI and MHCII, as well as T-cell costimulatory molecules such as CD86, to effectively stimulate CD8 and CD4 T-cell immunity. With respect to transfection of DCs, certain
1 Department of Chemical Engineering, Monash University, Clayton, Victoria, Australia; 2Department of Immunology, Central Clinical School, Faculty of Medicine, Nursing and Health Sciences, Monash University, Melbourne, Victoria, Australia and 3Department of Microbiology, Monash University, Clayton, Victoria, Australia. Correspondence: Dr C Selomulya, Department of Chemical Engineering, Monash University, Clayton Campus, Wellington Road, Clayton, Victoria 3800, Australia. E-mail: [email protected] 4 These authors contributed equally to this work. Received 16 April 2013; revised 15 October 2013; accepted 18 November 2013; published online 19 December 2013
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213 of targeting by combining magnetic nanoparticles, which initiate the surface of DCs under an external magnetic field, with targeting of specific DC receptors by HA ligands on the surface of the magnetic vectors. Here for the first time we report the results of utilizing the system of SPIONs/PEI/DNA-HA malaria gene complexes to target and transfect DC. Given the magnetic core, and the potential opportunity offered for directional transfection, we have further analyzed transfection efficacy with or without the added variable of an external magnetic field. In addition, we assessed the maturation/activation state of the DCs in vitro after interaction with these nanoparticles and magnetic fields. We focused on a quaternary system for blood-stage malaria DNA vaccine that encodes P. yoelii merozoite surface protein MSP1–19 (VR1020PyMSP1–19). The effects of combining different molecular weights of HA with different percentage charge ratios of HA: PEI on stability, cytotoxicity and DC transfection/maturation efficiency of the polyplexes were all investigated in vitro. This work illustrates how interactions of such well-designed quaternary complexes
plasmid DNA formulations or delivery systems may be able to target DCs for improved DNA uptake—for example, polyplexes with longer diblock copolymer chains showed the highest cellular uptake by DCs compared with short chain followed by effective gene transfection.10 Hyaluronic acid (HA) is a high-molecular-weight (HMW) mucopolysaccharide naturally present in all living organisms.11 It consists of a linear, unbranched, polyanions formed by D-glucuronic acid and N-acetyl-D-glucosamine repetitive units, proven to be low in toxicity, non-antigenic, non-immunogenic, biodegradable and biocompatible.11 Toll-like receptors (TLR) including TLR4 and TLR2, CD44, RHAMM, TNFIP6, HARE and LYVE-1 have been described as the main receptors for HA for different biological functions.12 A gene delivery system consisting of SPIONs/PEI/DNA-HA polyplexes can therefore be used to target DCs. The uptake of these polyplexes would be facilitated by HA receptor-mediated endocytosis on the surface of DCs, while at the same time maintaining the endosomal escape capacity of the PEI polymer.13 Such a system could provide an additional layer
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Figure 1. YFP expression and maturation of CD11c þ DCs induced by addition of functionalized DNA complexes to cells at a mass of 2 mg per well. DCs were isolated from the old C57BL/6 mice and incubated with different DNA complexes. After 24 h, cells were harvested and stained for expressions of CD86, MHC class I and MHC class II and analyzed by flow cytometry: (a) representative set of flow cytometry histograms of CD86 expression on CD11c þ DCs expressing YFP. Gray shaded area: untreated cells; (b) CD86 maturation markers on CD11c þ DCs expressing YFP; (c) YFP expression level and MHC I, MHC II costimulatory molecule expression on CD11c þ DCs. Data are analyzed statistically using one-way analysis of variance (ANOVA), and the comparison of means conducted using Tukey multiple comparison test. Results are expressed as means±s.d. (n ¼ 3). *Po0.05, **Po0.001, ***Po0.0001 (w magnet indicates application of an external magnetic field; w/o magnet indicates without application of an external magnetic field). & 2014 Macmillan Publishers Limited
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214 under magnetic field may markedly influence the delivery of genetic cargos to DCs. RESULTS The detailed characterization data of SPIONs/PEI/DNA-HA polyplexes as well as cell viability as measured by MTT (3-[4,5dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assays in COS-7 cells treated with these polyplexes can be found in Supplementary Information (Supplementary Figures S1–S6). The rationale of selecting this system has been discussed in our recent work.14 We examined the effects of magnetofection using quaternary polyplexes on mouse bone marrow-derived DC transfection and subsequent maturation (assessed by expression of MHCI, MHCII and CD86) after 5 h incubation with 2 h application of an external magnetic field. As shown in Supplementary Figure S6, the typical purity of CD11c þ DCs, which is the transmembrane protein found at high levels on most DCs, was B70–80%. Treatments with different DNA complexes, especially quaternary polyplexes, show no significant negative effects on the expression of CD11c on DCs compared with the normal control. Internalization of exogenous antigens is a prerequisite for APCs transfection with the antigen of interest and would improve their antigen presentation to T cells. Magnetofection-based DCs transfection and maturation in vitro Flow cytometry analysis results of yellow fluorescent protein (YFP) expression in CD11c þ DCs show that almost all SPIONs/PEI/ DNA-HA polyplexes increased significantly the transfection efficiency under the magnetic field, compared with transfection with other DNA complexes and even quaternary complexes without an applied magnetic field, regardless of the HA: PEI charge ratio or HA molecular weight (Figure 1). A trend was further observed between DC cellular uptake via magnetofection and increasing the molecular weight of HA in the quaternary polyplexes. Effect of HA blocking on cellular uptake of polyplexes with HA receptors Flow cytometric analysis results of YFP expression in CD11c þ DCs show that the DC pretreated with excess HA ligand has nearly 50% reduction in YFP gene expression compared with the transfection with cells pretreated with phosphate-buffered saline (PBS) alone (Figure 2a). Figure 2b shows that all wells treated with quaternary
HA-coated polyplexes induced activation of DC independently of CD44 We assessed the CD44 expresssion level on total CD11c þ DCs and CD11c þ DCs expressing YFP after treatment with different gene complexes compared with CD44 expression level in untreated CD11c þ DCs. Our results show strong expression of surface CD44 receptor around 90% in all CD11c þ DCs indicating the presence of this type of receptor in our DC cultures (Figure 3).
DISCUSSION DCs transfection and maturation An important concern resulting in insufficient gene delivery of nonviral gene vectors in vitro is the low accumulation of gene complexes at the cell surface. Here we applied an external magnetic field on the quaternary magnetic gene vector to accumulate the gene vector on DCs in the culture media.15,16 We used DNA encoding a fluorescent protein (YFP) mixed in 1:1 ratio with the MSP1–19 expressing vector to allow us to read out the efficiency of DNA uptake. Western blot analysis using antiPyMSP1–19 antibodies was further performed on transfected DCs pellet to identify the identity of recombinant proteins produced by PyMSP1–19 plasmid in CD11c þ DCs. The result shows that the expressed protein in DCs is equal to the PyMSP1–19 protein expressed in Escherichia coli (Supplementary Figure S7). The ability of DCs to drive antigen-specific immunity is dependent on their degree of maturation. Flow cytometric analyses provided evidence that generally, the treatment of DCs with plasmid DNA or even plasmid DNA loaded onto nanoparticles in general enhanced the expression of CD86 in CD11c þ DCs expressing YFP. This is because bacterial plasmid DNA possesses inherent immune adjuvant activity based on its unmethylated CpG motifs in the plasmid DNA backbone, previously shown to act as a potent DNA vaccine adjuvant in rodents.17 Culturing DCs with quaternary polyplexes induced substantial upregulation of CD86 in the overall culture, it specifically increased both efficiency of YFP gene transfection in CD11c þ DCs and concomitant maturation through upregulated expression of costimulatory molecule CD86 and MHC molecules (Figures 1b and c).
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polyplexes pretreated with PBS alone (without HA) upregulated CD86 maturation marker on CD11c þ DCs expressing YFP regardless of the HA molecular weight, in contrast to pretreated HA wells.
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Figure 2. Effects of HA-pre-addition on the YFP expression and costimulatory surface marker (CD86) on CD11c þ DCs mediated by quaternary polyplexes: (a) the percentage of total CD11c þ DC cells expressing YFP; (b) the percentage of CD11c þ DCs expressing YFP with upregulation of CD86 maturation signal (n ¼ 3). ***Po0.0001 (w magnet indicates application of an external magnetic field; w/o magnet indicates without application of an external magnetic field). Gene Therapy (2014) 212 – 218
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Figure 3. Effects of different DNA complexes on the expression of CD44 receptors on CD11c þ DCs population: (a) the percentage of total CD11c þ DC cells; (b) the percentage of CD11c þ DCs expressing YFP. Data are analyzed statistically using one-way ANOVA, and the comparison of means was conducted using the Tukey multiple comparison test. Results are expressed as means ±s.d. (n ¼ 3). ***Po0.001 (w magnet indicates application of an external magnetic field; w/o magnet indicates without application of an external magnetic field).
In contrast, DCs treatment with different DNA complexes excluding quaternary polyplexes did not significantly enhance YFP gene expression and also the level of stimulatory markers (CD86, MHC molecules) on the CD11c þ DCs expressing YFP, suggesting that internalization is the main contributor in quaternary polyplexes-induced concomitant DC gene transfection and maturation (Figures 1a and b). The YFP gene expression and phenotypical maturation signals observed during DCs exposure to quaternary polyplexes were much higher compared with those of ternary polyplexes. These results support the notion that factors such as endocytosis via HA receptors may have a role in DNA complexes-induced DC maturation, as HA was present in the quaternary system. In addition, unmethylated CpG motifs of plasmid DNA are known to be recognized by the TLR9 in DCs which have an important role in long-term vaccination protocols.18 It is likely that upon endocytosis, plasmid DNA becomes at least partially degraded by DNase II in the endosomal-lysosomal compartment to release CpG motifs. As TLR9 is not present on the surface of the cells, targeted delivery of plasmid DNA by means of nanoparticles to the endosomal compartment might be important for potential association of DNA with the TLR9 receptor in the endosome and trigger DC maturation followed by the immune response. The effects of polyplexes gene transfection on DCs transfection and maturation in the in vitro culture correlated with the extent of in vivo DC maturation through the consequent adjuvant effect on the immune response toward the antigen of interest. The data obtained highlighted (for the first time) to the direct interactions of DC with magnetic gene complexes coated by PEI and HA polymers in promoting DCs maturation. Therefore, suitable surface modifications by HA should allow specific endocytosis of gene complexes, to affect the pathways of intracellular processing and trafficking. It also suggests that the prime mechanism by which the gene complexes enhance an adaptive immune response is maturation of the APCs, specifically DC, such that they become efficient at antigen presentation and T-cell stimulation to generate an immune response to the antigen of interest. As these results suggest that quaternary polyplexes were stimuli of DC maturation through internalization, probably by specific HA receptor, we sought to further investigate the effects of HA molecular weight and magnetic field applications on the uptake of quaternary polyplexes by murine DCs, to determine to what extent the particle endocytosis had a role in DC maturation. As shown in Figures 1a and b, SPIONs/PEI/DNA-HA polyplexes at HMW HA and 100% charge ratio of HA: PEI (100% HMW polyplexes) under magnetic field display the highest YFP gene & 2014 Macmillan Publishers Limited
expression, accompanied also by maturation through upregulated expression of CD86, compared with transfection with other complexes. The efficiency is much higher than DNA alone (Po0.0001). Although SPIONs/PEI/DNA ternary polyplexesmediated transfection was also done under a magnetic field, they showed significantly lower transfection efficiency than SPIONs/ PEI/DNA-HA quaternary polyplexes and DNA-Lipofectamine 2000 (a standard gene delivery agent) (Po0.05). Interestingly, 100% HMW polyplexes show approximately the same levels of YFP and CD86 expression under or in the absence of magnetic field. The physicochemical properties of HA are related to its molecular weight.19 Higher molecular weight is associated with longer molecular chains that in turn increase the mucoadhesive properties of HA in solution. Lim et al.20 also found that the incorporation of HA with molecular weight of 8.5 105 Da into macro-particles increased mucoadhesive properties more than chitosan-based systems. HMW HA in quaternary polyplexes is likely to increase the mucoadhesive and viscoelastic properties of the complexes, accordingly enhances large molecular chains of HA ligands accessibility to the DC receptors and promotes the multivalent binding to receptors,21 regardless of the presence or absence of the magnetic field. The efficiency of HA polymer with HMW in accessing DC cells is associated with their ability to bind to DCs receptors, with a previous study showing that HMW HA binding was irreversible to CD44 cell receptors, although the binding became weaker and reversible with decreased HA molecular weight.22 On the other hand, applying an external magnetic field could enhance the accumulation and attachment of low molecular weight (LMW) quaternary polyplexes on the surface of DCs, resulting in significant increases of the cellular uptake of the complex (Po0.001), accompanied by improved phenotypic maturation of DCs.23 Indeed, the effects of magnetic field on LMW (LMW HA) quaternary polyplexes are more pronounced in the 5% LMW polyplexes (Figure 1b). This new observation is in good agreement with a previous study by Qhattal et al.24 showing that DC cellular targeting efficiency of HA-liposomes nanoparticles depended strongly upon HA molecular weight and grafting density, with HMW HA resulting in better cellular adhesion and internalization of HA-liposomes to DCs. In this study, the concentration of HA is in the range of 0.012–0.14 mg ml 1, approximately similar to the range used by Do et al.25 who found that the addition of 0.01–1 mg ml 1 of HA in DCs culture were very effective in growing cells, whereas high HA concentration of 0.5 mg ml 1 inhibited the growth of DCs. In agreement with our data, the polyplexes with these Gene Therapy (2014) 212 – 218
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216 concentrations could interact properly with DCs and promote their maturation. In addition, our goal was to determine whether HA-coated quaternary polyplexes could increase the functional uptake of DNA by DCs for targeted gene expression and antigen presentation. After DC transfection, the produced antigen was processed and presented by MHC class I and MHC class II to stimulate cytotoxic T-cells and helper T cells, respectively.26 These intracellular produced antigens by DCs are hard to detect and present as MHC class I and II, as the molecular endocytosis of antigen is not necessarily sufficient to ensure efficient antigen presentation.27 However, we showed that the quaternary polyplexes considerably enhanced antigen uptake and presentation by DCs, probably via HA-mediated endocytosis that has been enhanced by the application of external magnetic field in vitro (as shown in Figure 1c) and via initial in vivo studies (Supplementary Figure S8). The use of quaternary polyplexes under magnetic field significantly upregulated MHC class I and MHC class II expressions on DCs, supporting further their advanced maturation profile (Figure 1c). HA molecules may also act as a condensation agent for DNA in a similar manner to PEI, especially as DNA molecules were mixed with HA polymer before addition to SPION-PEI complexes. Kim et al.28 showed by gel electrophoresis that DNA could interact to some degree with HA fragments, with only small portions of the DNA molecules able to be released from the DNA-HA matrix. It is also known that the positively charged PEI condenses DNA into a compact colloid while affording a good protection from DNase digestion.13 Thus, the interaction of DNA with HA and then condensation together with PEI-coated SPIONs could well increase the stability of DNA in the complexes, and at the same time protect DNA from DNase degradation. However, strong interactions between PEI and DNA may delay DNA release from complexes or even prevent all DNA reaching the nucleus, leading to a decrease in transfection efficiency and gene expression. HA might be useful in this regard as well, behaving here like a transcriptional activator by lessening the electrostatic complexation between DNA and PEI polymer (refer to Supplementary Information and Supplementary Figure S4A, gel retardation assay), thus facilitating the interaction of transcription factors with plasmid DNA. Granulocyte macrophage colony stimulating factor (GM-CSF) was added at a final concentration of 10 ng ml 1 to the cell culture in order to stimulate DC differentiation. Adding this cytokine to DC culture has been described previously to activate DCs proliferation and cellular uptake.29 Adding this cytokine to the cell culture with the quaternary polyplexes might promote cellular uptake of the gene complexes, followed by gene expression in the DCs. Our study result seemed to confirm the finding by Ali et al.30 in suggesting that placing DC in the media containing low levels of GM-CSF (50 ng ml 1) during PEI-DNA gene transfection could greatly enhance gene transfection, comparatively to high amounts of GM-CSF (500 ng ml 1) that could promote the degradation of intercellular DNA and its protein products. Modifying the magnetic gene vector with HA polymer also led to higher stability of complexes in the DCs media. This could lead to more cellular uptake of the complexes with well-timed dissociation of the DNA–polymer complex. Other possible mechanisms of high gene expression relate to the presence of HA, was the notably decreased cytotoxicity and the size of quaternary polyplexes in the cell media to around 150 nm at the optimal charge ratio of 100%, leading to better distribution with high cellular uptake.31 Effect of HA blocking on cellular uptake The YFP gene expression in CD11c þ DCs treated with HMW polyplexes without pretreated HA showed significantly the highest gene expression (Po0.0001), whereas LMW HA polyplexes Gene Therapy (2014) 212 – 218
with and without pretreated HA did not show any significant difference in gene expression (Figure 2a). This suggested that the YFP expression of HMW polyplexes could be mediated by HMW HA-specific receptors on the surface of DC cells, more than the LMW HA that could be competitively inhibited by excess free HA. The trend shown in Figure 2b is in agreement with Ito et al.,32 who showed that the CHO cells pretreated by HA had diminished gene expression compared with highly enhanced luciferase gene expression of HA-coated ternary complexes (DNA/PE/HA) pretreated with only PBS. Taken together, these results suggested interactions between HA special receptors on DC specifically with HMW HA-coated polyplexes that could encourage uptake via these receptors.32 Activation of DC independently of CD44 CD44 is a principal cellular HA receptor,12,33 raising the question on whether CD44 could also mediates the HA-coated quaternary polyplexes uptake. In general, untreated or treated CD11c þ DCs with different DNA polyplexes showed approximately the same percentage of CD44 expression. The ternary polyplexes, DNA/Lipofectamine complexes and even free DNA slightly, but not significantly, upregulated CD44 expression level on DCs especially with cells expressed YFP compared with control DCs. In contrast, DC pretreated with high concentration of HA before adding the quaternary polyplexes showed significant trends toward decreasing CD44 in total DCs or DCs expressed YFP compared with control DCs (Figures 3a and b). Adding HA at high concentration may potentially inhibit CD44 expression, as previous study of Kaya et al.34 suggested that the abnormal accumulation of hyaluronate in superficial dermis due to some medical reasons was accompanied by the decline in CD44 expression in the keratinocytes, and it could be argued that a similar mechanism could promote low CD44 expression on DCs when pretreated with high amounts of HA (Figure 3). A recent report showed that under normal condition, CD44 receptors are rapidly recycled back from the intracellular compartment to the plasma membrane following internalization and release of cargos.35 Therefore, the possibility of a lower expression of CD44 due to uptake of the HA-coated quaternary polyplexes as a receptor for HA could be excluded, as the fluorescence-activated cell sorting analysis was performed 24 h after adding the polyplexes in the cell culture, allowing enough time for CD44 to recycle back to the cell surface. The transfection study (Figure 1) shows that DCs treatment with the quaternary polyplexes especially under magnetic field displays significantly high levels of YFP gene expression accompanied by significant upregulation in the CD86 expression; thus, implying that quaternary polyplexes were actively uptaken by DCs via different type of HA receptors than CD44. Most interestingly, there is no significant difference in CD44 expression levels between total CD11c þ DCs and CD11c þ DCs transfected with YFP gene for cells treated with different HA-coated quaternary polyplexes compared with control DCs. Taken together, these data potentially demonstrate that CD44 receptors were not actively involved in HA quaternary polyplex uptake by DCs, thereby suggesting a possible role for other receptors of HA, such as TLR2 and TLR4, CD38, LYVE-1 and RHAMM or even unknown novel receptors.12 Others have suggested that the mammalian endocytic HA receptor is HARE instead of CD44.36 Furthermore, Do et al.25 previously reported that HA could activate DC through CD44-independent pathway, although CD44 on antigen-specific T-cells had a critical role during interaction with DC to present antigen and subsequently T-cells expansion. Moreover, Termeer et al.37 found that CD44KO-derived DCs (knockout mice) showed similar upregulation of costimulatory molecules like wild-type-derived DCs after HA treatment. It should be noted that these studies were different from the current study in that they primarily investigated the ability of HA-treated DCs to & 2014 Macmillan Publishers Limited
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217 activate alloreactive T-cells. Our aim here was to transfect DCs using malaria DNA vaccine condensed on HA-coated magnetic quaternary polyplexes and at the same time to increase DCs maturation through upregulation of costimulatory molecules. HA on the surface of these quaternary complexes could act as ligands to enhance receptor-mediated endocytosis through specific HA receptors on DCs. However, we cannot eliminate the possibility that CD44 could make a minor contribution to the cellular uptake of quaternary polyplexes involved in the polyplexes transfection and maturation of wild-type-derived DCs. As we did not detect expressions by other receptors in mature or immature DC by reverse transcriptase-PCR, we could exclude the effects of other HA receptors. Further study will be necessary to determine the specific type of DCs receptor contributing to the uptake of quaternary polyplexes. Although it is reported in the literature that DC is problematic to transfect, here we demonstrate that quaternary magnetic polyplexes containing HA could be used to transfect DCs in vitro highly effectively with a model DNA-based malaria vaccine candidate. The transfection could be enhanced by increasing the HA molecular weight and the application of an external magnetic field. The size of HA ligand on the polyplexes was an important determinant of its cellular uptake, with higher molecular weight of HA resulting in better cellular adhesion and internalization. Moreover, the presence of HA loosened the strong complexation between DNA and PEI polymer, but did not result in DNA disassembly, with higher cell viability compared with those treated without HA in the polyplexes. These quaternary polyplexes also had adjuvant activity that encouraged DC maturation (specifically for the cells that had internalized the complexes), while the application of an external magnetic field improved gene delivery to DCs at low DNA doses, while also upregulating DC maturation markers. This new information will be crucial for the design of DNA gene delivery tool to somatic cells such as DCs for potent immune activation, with further studies currently underway to investigate the detailed uptake mechanisms and also the specific type of DCs receptor of this gene delivery system both in vitro and in vivo.
Supplementary Information. Bone marrow-derived DCs were generated as previously described with minor alterations.38
Magnetofection-based DC transfection and maturation in vitro To examine the effects of quaternary polyplexes on DCs transfection and maturation state, on day 5 of the bone marrow-derived DCs culture, culture plates were centrifuged and the medium was removed. The cells in each well were washed with serum-free RPMI 1640 medium. Freshly prepared ternary and quaternary polyplexes of SPIONs/PEI/DNA-HA with two different molecular weights of HA and two different ratios of HA: PEI (charge) were diluted in serum-free RPMI 1640 medium for 15 min at room temperature. The polyplexes with N/P of 10 were then added to the cells with a fixed amount of MSP1–19/YFP plasmid mixtures of 2 mg per well. The cells were cultured with a neodymium-iron-boron magnet positioned under each well for 2 h. After 5 h, the culture media containing polyplexes were removed from each well and replaced with 2 ml of the fresh complete medium containing 10 ng ml 1 (GM-CSF) and cultured for a further 24 h in 37 1C incubator with 5% CO2. DCs transfection efficiency by quaternary polyplexes was examined with or without the application of magnetic field during the process. Transfection of DCs with plasmid DNA alone without complexation in the polyplexes was also examined for comparison. To examine the transfection efficiency of quaternary polyplexes in DCs and maturation of CD11c þ DCs, the non-adherent cells were harvested and washed with PBS after 24 h. The cells were stained using the following antibodies: APC-CD11c, biotinylated-CD86, APC-CD44, PE-MHC Class I, APC-Cy7-MHC Class II, whereas dead cells were excluded using live/dead fixable dead cell stain kit (Aqua LIVE/DEAD, Invitrogen). Biotin-labeled monoclonal antibodies were developed with PE-Cy7-streptavidin and analyzed by flow cytometry (FACSCalibur). Untreated DCs were also included as negative controls. Quaternary polyplexes coated with HA can be easily dispersed in cell medium, favorably responding to receptor-ligand interactions with HA receptors positive-cells such as DCs, although they could also loosen strong electrostatic interactions between DNA and PEI (Supplementary Figure S4A). A way to prove the existence of these interactions was by deliberately blocking HA receptors on the cells by pretreatment with a saturable amount of free HA prior incubation with different plasmid complexes. Before adding different DNA polyplexes to the cells, 200 ml of HA solutions (3 mg ml 1 in PBS) (HMW or LMW) were added to DC cells in addition to 1 ml of RPMI medium. After incubation for 60 min at 37 1C, the cells were washed with RPMI media to remove unbound HA and then different complexes were added to the wells by application of external magnetic field for 2 h, with similar treatment as previously referred to in the transfection assay.
MATERIALS AND METHODS Materials Sodium hyaluronate (HA) with average molecular weight of o10 and 900 kDa were purchased from Life Core Biomedical LLC (Chaska, MN, USA). RPMI 1640 medium, 0.05% trypsin-EDTA, penicillin/streptomycin, L-glutamine, Lipofectamine and fetal calf serum 2000 were from GibcoBRL (Gathersburg, MD, USA). Hamster-anti-mouse APC-CD11c antibody, rat-anti-mouse biotinylated-CD86 (B7.2), rat-anti-mouse APC-CD44 and ratanti-mouse PE-MHC Class I were purchased from BD Pharmingen (Franklin Lakes, NJ, USA), whereas GM-CSF were ordered from PeproTech (Rocky Hill, NJ, USA). PE-Cy7-streptavidin was from BD Bioscience (Franklin Lakes, NJ, USA), whereas rat-anti-Mouse APC-Cy7-MHC Class II was from eBioscience (San Diego, CA, USA). Live/dead fixable dead cell stain kit (Aqua LIVE/DEAD; Invitrogen (Carlsbad, CA, USA)) and Lipofectamine 2000 (Gibco-BRL) were supplied by Invitrogen. Mammalian expression vector VR1020 (Vical Inc., San Diego, CA, USA), VR1020-PyMSP1–19 and VR1020-YFP plasmids were obtained from collaborators at the Coppel lab (Department of Microbiology, Monash University, Clayton, Victoria, Australia). C57BL/6 mice (aged 6–8 weeks) were supplied by the animal facilities in the Alfred Medical Research and Education Precinct (AMREP). Arrays of permanent magnets of neodymium-iron-boron (Nd-Fe-B) in the format of a six-well plate were used for in vitro transfection experiments. The magnets were circular disk Nd-Fe-B magnets (diameter 25 mm, height 5 mm) glued onto the bottom of a six-well plate. Detailed procedures for preparation of SPIONs/PEI and SPIONs/PEI/ DNA-HA quaternary polyplexes, purification of plasmids DNA, characterization of these polyplexes via size and zeta potential measurements, DNA retardation and DNase I sensitivity assays and the determination of COS-7 cell variability by MTT colorimetric assay can be found in the & 2014 Macmillan Publishers Limited
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We acknowledge Dr Tim Williams from the Monash Centre of Electron Microscopy for assistance in TEM imaging.
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Supplementary Information accompanies this paper on Gene Therapy website (http://www.nature.com/gt)
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