2016, Vol. 27 
b Department of Physical Sciences, Charleston Southern University, Charleston, SC 29423, United States
Poly(amidoamine) (PAMAM) dendrimers are hyperbranched polymers with high molecular uniformity, narrow molecular distribution, well defined size and structures, and multifunctional terminal surface [1]. PAMAM dendrimers have found a wide range of applications in biomedical field including cancer targeting [2, 3], drug delivery [4, 5], gene delivery [6, 7], and imaging [8, 9] due to their nano-sized structures, water solubility and biocompatibility. Previously, our research group have used the synthetic PAMAM dendrimer conjugated to a natural polymer, hyaluronic acid (HA), to form a fast cross-linking hydrogel which could support the growth of bone marrow stem cells (BMSCs) in a 3D environment. In addition, we found that the introduction of an Arg-Gly-Asp (RGD) peptide into the hydrogel system could significantly improve the cell viability, proliferation, and attachment. In this work, we extend our previous study by incorporation of different adhesive peptides that have been reported for promoting cell responses [10-12] to PAMAM dendrimers. The functionalized PAMAM dendrimers were used as supporting substrates for enhancing cell responses in vitro. Besides BMSCs, a common cell source for tissue engineering [13-15], pheochromocytoma (PC12) cells were selected as a model of neuronal proliferation and differentiation due to their ability to differentiate and produce neurites in response to nerve growth factor (NGF) [16] and insoluble cues such as extracellular matrix ligands [17].
Herein, PAMAM dendrimers of generation 5 were first modified and functionalized with divinyl sulfone (DVS) to establish vinyl sulfone end groups. Then, they were conjugated to Arg-Gly-Asp (RGD), Tyr-Ile-Gly-Ser-Arg (YIGSR), or Ile-Lys-Val-Ala-Val (IKVAV) peptides [18-21]. The structures of the peptide modified PAMAM are characterized using nuclear magnetic resonance (1H NMR) and matrix-assisted laser desorption ionization-time of flight (MALDITOF). Cell adhesion of BMSCs on the functionalized PAMAM coated substrate was analyzed using centrifugal adhesion assay and light microscopy [22]. NGF-induced differentiation and neurite growth of PC12 cells were determined using light microscopy and computer-based quantitative image analysis. Moreover, cell viability and proliferation of differentiated PC12 cells were evaluated using CellTiter-Blue® (CTB) assay and live cell staining. We reported the potential of functionalized PAMAM dendrimer as a polymeric biomaterial for tissue engineering applications.
2. ExperimentalPAMAM dendrimer of generation 5 (PAMAM G5) was ordered from Dendritech Inc. (Midland, MI, USA). Characterization by potentiometric titration revealed that there were 110 primary amine groups on the surface of the G5 dendrimer. The CGRGDS peptide and laminin peptides IKVAVC and YIGSRC were synthesized by Peptide 2.0 Inc. (Chantilly, VA, USA) with the structure confirmed by mass spectroscopy. Other chemicals and solvents were ordered from Aldrich and were used as received. PAMAM G5 was surface modified by partial acetylation, further reaction with divinyl sulfone (DVS), and cross-linking with the peptides bearing a terminal cysteine through a thiol-ene reaction (dendrimer 5, 6, and 7). 1H NMR spectra of dendrimer species were recorded using a Varian 400 MHz spectrometer (Santa Clara, CA, USA) in D2O solvent. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) spectra of dendrimer species were also recorded using Ultraflex TOF/TOF (Bruker Daltonics) MALDI time-of-flight mass spectrometer.
BMSCs were isolated from the bone marrow of young adult 160-180 g male Sprague-Dawley rats (Charles River Laboratories) as described in our previous reports [23, 24]. The cells were passaged no more than seven times after isolation and maintained in complete primary media [Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS)], kept at 37 ℃ in a CO2 incubator with 5% CO2:95% air. Centrifugal adhesion assay of BMSCs on the functionalized PAMAM coated substrates was performed following the literature protocol [22].
PC12 cells, a rat adrenal pheochromocytoma cell line that is induced by neurite growth factor (NGF) into a neuronal phenotype, were purchased from ATCC (Manassas, VA). The cells were designated as received at passage 1 and used at passage 4-5. PC12 cells were maintained on 75 cm2 tissue culture-treated polystyrene plates (Corning) coated with type Ⅳ collagen (Sigma) and RPMI-1640 medium (Sigma) supplemented with 10% horse serum (Sigma), 5% FBS (Atlanta biologicals), 1×Penicillin-Streptomycin-Amphotericin B (from MP Biomedicals, 100 U/mL penicillin and 1000 U/mL streptomycin solution, 0.25 mg/mL amphotericin B), kept at 37 ℃ in a CO2 incubator with 5% CO2:95% air modified from the literature protocols [25, 26]. PC12 neurite outgrowth on the functionalized PAMAM coated substrates were accessed by using inverted phase contrast microscope and ImageJ. Cell viability and proliferation of PC12 were also studied using CellTiter-Blue® (CTB) assay (Promega) and live cell staining.
The details of all methods and any associated references are available in the electronic supplementary information.
3. Results and discussionOne of current paradigms for designing biomaterials in biomedical and tissue engineering applications is to provide materials with biofunctionalities to facilitate the cell-material interaction [27-29]. In this study, we demonstrate that PAMAM dendrimer, a synthetic biomaterial, could be successfully modified with different biofunctionalities in a well-defined manner. Three peptides, including RGD, YIGSR, and IKVAV, were selected to conjugate to PAMAM dendrimers because they represent the celladhesive domains of fibronectin, collagen and laminin [30]. They have been used in a number of applications to induce specific cell behaviors [21, 31-33].
The chemical synthesis of PAMAM dendrimer conjugates is displayed in Scheme 1. The commercial PAMAM dendrimer G5 bearing 110 terminal amine groups was used as a template. The number outside the parenthesis of each functionality in subscript denotes the number of that functional group intended to be attached per dendrimer, which can be varied and chemically optimized depending on the property required for specific applications. The detailed description of synthesis has been reported in our previous publication [34]. In brief, PAMAM G5 was surface modified by partial acetylation to yield G5-Ac(75) (dendrimer 2) with 75 terminal amine groups acetylated. The remaining 35 amine groups on dendrimer were reacted with Traut’s reagent to afford the thiol terminated dendrimer G5-Ac(75)-SH(35) (dendrimer 3), which was further reacted with divinyl sulfone (DVS) to establish multiple vinyl sulfone end groups for cross-linking with the peptides bearing a terminal cysteine through a thiol-ene reaction (dendrimer 4). Finally, the peptide CGRGDS was attached to the vinyl sulfone group of dendrimer 4 to achieve the dendrimer product G5-Ac(75)-DVS(34)-RGD(1) (dendrimer 5). Laminin peptide functionalized dendrimers (6 and 7) were synthesized with the same procedure as for dendrimer 5 by reaction of dendrimer 4 to peptides YIGSR and IKVIV with each peptide bearing a terminal cysteine for covalent conjugation on dendrimer. The dendrimer product for each synthetic step was purified by membrane filtration through a 10, 000 MWCO membrane and extensively washed with PBS buffer and water, followed by lyophilization.
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| Scheme. 1. Synthesis of functionalized PAMAM dendrimer | |
1H NMR was used to characterize the chemical structures of the dendrimer species. The 1H NMR spectra of dendrimers 3 through 5 have been reported in our previous publication [34], demonstrating the successful sequential attachment of each group on dendrimer. Here, the 1H NMR spectra of dendrimers 1 through 7 were presented (Figs. S1 and S2 in Supporting information). The results confirmed that dendrimer 2 through 4 were successfully created by each step of the reaction. However, not many distinctive RGD, YIGSR, and IKVAV peaks could be observed because only one equivalent of each peptide was conjugated to dendrimer 4 in order to maintain the solubility of peptide functionalized dendrimers 5-7. Second, most peptide peaks were aliphatic and buried under dendrimer peaks, which made the peptide peaks not observable under or among dendrimer peaks.
Alternatively, the number of RGD, YIGSR, and IKVAV attached were determined by MALDI-TOF, which has been demonstrated in our previous study [34]. Figs. S3 and S4 displayed the MALDI-TOF analysis of dendrimers 1 through 7 with average molecular weights labeled for each species. With the molecular weight increase of each step, the success of sequential conjugation of molecules to dendrimer was confirmed and the number of molecules attached to dendrimer could be quantitatively determined. The summary of molecular weights and number of functional groups per dendrimer determined by 1H NMR and MALDI-TOF were shown in Table S1 in Supporting information.
The peptide-modified PAMAM dendrimers were used as coating materials for cell culturing. The peptide-modified PAMAM coated substrates were created by simply incubating the tissuecultured well plates with the functionalized PAMAM dendrimer in PBS at concentration of 1% w/v for 1 h, allowing the hydrophilic interaction between the functionalized PAMAM and the surface of the well plates. To examine the strength of adhesion of BMSCs to the peptide-modified PAMAM coated surfaces, the cells were seeded on the peptide-modified PAMAM coated substrates and incubated for 1, and 2 h. The surfaces then were subjected to an inverted spin at 10 g (after 1 h) and 30 g (after 2 h) for 5 min. The percent of cells remaining on the surfaces was determined. As seen in Fig. 1A, there was a greater amount of early cell attachment to surfaces coated with the three adhesive peptide-modified PAMAM (dendrimer 5, 6, and 7) than there was to the control surfaces. After 1 h of incubation and exposure to inverted-spin, greater than 45% of the cells remained adherent to all of the peptide-modified PAMAM coated surfaces, which was significantly higher than the percentage of cells remaining on the control surfaces (Fig. 1B). Similarly, more than 95% of the cells remaining on all of the peptide-modified PAMAM coated surfaces after 2 h of incubation, which was higher that the percentage of cells remaining on the control surfaces. The results were consistent to the study of Saneinejad et al., who have shown that primary neural cells adhere preferentially to, RGD, YIGSR, or IKVAV modified glass surfaces over PEG modified glass surfaces [35]. Similar to the study in vascular smooth muscle cells, the cells were more strongly adhered to surfaces modified with adhesive peptides than to control surfaces [36]. It also suggested that the adhesive peptides were successfully conjugated to PAMAM dendrimer to promote cell-material interaction and subsequently cell attachment.
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| Figure 1. Centrifugal adhesion assay to measure detachment forces for BMSCs incubated on substrates coated with native PAMAM carrying DVS (dendrimer 4), RGD modified PAMAM (dendrimer 5), YIGSR modified PAMAM (dendrimer 6), or IKVAV modified PAMAM (dendrimer 7). (A) Inverted phase contrast micrographs of BMSCs prior and postcentrifugation. (Right) The images of BMSCs after 1 h of incubation, pre-and post-inverted spin at 10 g (~1.2 pN/cell) for 5 min. (Left) The images of BMSCs after 2 h of incubation, pre-and post-inverted spin at 30 g (~3.6 pN/cell) for 5 min. (B) Percentage of remaining cells after 1 h of incubation and an inverted spin at 10 g (~1.2 pN/cell) for 5 min. (C) Percentage of remaining cells after 2 h of incubation and an inverted spin at 30 g (~3.6 pN/cell) for 5 min. The values expressed are means (n=4)±SD. Samples were compared using two-tailed equal variance Student t tests, ** P < 0.005, *** P < 0.0005. | |
To examine the ability of the peptides-modified PAMAM coated substrates on differentiation and proliferation of PC12 cells, the cells were cultured with NGF supplemented complete growth medium for 1 day, 2 and 4 days. As shown in Fig. 2A, the neurite outgrowth of PC12 cells was evident on all of the peptides-modified PAMAM coated substrates by 1 day and extensively promoted along the cultured period. Quantitative image analysis of the neurite lengths on all of the peptidemodified PAMAM surfaces were in range of 18-20, 50-60, and 90-115 μm on day 1, 2 and 4, respectively, which were significantly higher than the neurite length on the control surfaces (Fig. 2B). Similarly, the percent of cells expressing neurites on the peptidesmodified PAMAM surfaces significantly increased compared to the control surfaces. Namely, greater than 40% and 90% of the cells differentiated on the peptides-modified PAMAM surfaces after day 1 and 2, while only 10% and 50% of the cells differentiated on the control surfaces (Fig. 2C). In agreement with our findings, Ranieri et al. and Bellamkonda et al. showed that polymer surfaces coated by IKVAV peptides localize PC12 cell attachment and promote their neurite outgrowths [37, 38]. Moreover, PC12 cells have been shown to attach to RGDS, IKVAV, and YIGSR modified PEG, but not on non-adhesive modified PEG [39].
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| Figure 2. Neurite outgrowth analysis of PC12 cells cultured on substrates coated with native PAMAM carrying DVS (dendrimer 4), RGD modified PAMAM (dendrimer 5), YIGSR modified PAMAM (dendrimer 6), and IKVAV modified PAMAM (dendrimer 7) in NGF supplemented culture medium for 1 day, 2 and 4 days. (A) Inverted phase contrast micrographs of different PAMAM dendrimer matrices at different time points. All images share the same scale bar: 50 mm. (B) Average neurite length of PC12 cells on different PAMAM dendrimer matrices at different time points. (C) Percentage of cells displaying neurite outgrowths on different PAMAM dendrimer matrices at different time points. The values expressed are means (n=4)±SD. Samples were compared using two-tailed equal variance Student t tests, * P < 0.05, ** P < 0.005, *** P < 0.0005. | |
Aside from the neurite differentiation analysis, the CTB assay for the proliferation of differentiated PC12 cells was conducted after 2, and 4 days of culturing (Fig. 3). The results showed that all of the PAMAM coated substrates supported the proliferation of differentiated PC12 cells. Specifically, the RGD modified PAMAM coated substrates significantly promoted the proliferation of differentiated PC12 cells compared to other types of PAMAM coated substrates. Live cell staining images also confirmed the viability and proliferation of the differentiated PC12 cells. As shown in Fig. S5 in Supporting information, the cells were stained green [40, 41], indicating that the differentiated PC12 cells were viable on all of the PAMAM coated substrates upon the incubation. Additionally, the number of viable cells on all of the PAMAM coated substrates obviously increased on day 4 compared to day 2, indicating that PC12 cells were proliferated well on all substrates. Based on all of the cell study results, the RGD, YIGSR, or IKVAV-modified PAMAM dendrimer was an effective polymeric material to support and promote cell responses, namely cell viability, attachment, proliferation, and differentiation in the similar manners.
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| Figure 3. CTB® cell viability assay of differentiated PC12 cells cultured on substrates coated with native PAMAM carrying DVS (dendrimer 4), RGD modified PAMAM (dendrimer 5), YIGSR modified PAMAM (dendrimer 6), or IKVAV modified PAMAM (dendrimer 7) in NGF supplemented culture medium for 2 and 4 days. The values expressed are means (n=3)±SD. Samples were compared using two-tailed equal variance Student t tests, * P < 0.05. | |
4. Conclusion
PAMAM dendrimer has been successfully conjugated to reported adhesive peptides via multiple vinyl sulfone groups on dendrimer and thiol groups on adhesive peptides. Conjugation of RGD, YIGSR, and IKVAV peptides to PAMAM dendrimer significantly improved cell attachment of BMSCs, and neurite growth and differentiation of PC12 cells on 2D culture. We envision that this adhesive peptide conjugated PAMAM dendrimer could be a promising platform for biomedical and tissue engineering applications.
AcknowledgmentsThis research is financially supported by the NSF-ECCS 1509760, and NSF EPSCoR RII Track 1 cooperative agreement awarded to the University of South Carolina (NSF EPSCoR Cooperative Agreement No. EPS-0903795).
Appendix A. Supplementary dataSupplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2016.03.012.
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