Article pubs.acs.org/Langmuir
Altering Colloidal Surface Functionalization Using DNA Encapsulated Inside Monodisperse Gelatin Microsphere Templates James O. Hardin,† Alberto Fernandez-Nieves,‡,§ Carlos J. Martinez,⊥ and Valeria T. Milam†,§,∥,* †
School of Materials Science and Engineering, ‡School of Physics, §Parker H. Petit Institute for Bioengineering and Bioscience, and Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30032-0245, United States ⊥ School of Materials Engineering, Purdue University, West Lafayette, Indiana 47907-2045, United States ∥
S Supporting Information *
ABSTRACT: Soluble oligonucleotides are typically introduced to bulk solution to promote hybridization activity on DNA-functionalized surfaces. Here, an alternative approach is explored by encapsulating secondary target strands inside semipermeable colloidal satellite assemblies, then triggering their release at 37 °C for subsequent surface hybridization activity. To prepare DNA-loaded satellite assemblies, uniform gelatin microspheres are fabricated using microfluidics, loaded with 15 base-long secondary DNA targets, capped with a polyelectrolyte bilayer, and finally coated with a monolayer of polystyrene microspheres functionalized with duplexes comprised of immobilized probes and soluble, 13 base-long hybridization partner strands. Once warmed to 37 °C, secondary DNA targets are released from the gelatin template and then competitively displace the shorter, original hybridization partners on the polystyrene microspheres.
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INTRODUCTION Gelatin is a collagen-derived polypeptide with numerous healthrelated applications ranging from food to pharmaceuticals.1,2 Gelatin microspheres (GMS) have reportedly low immunogenicity, but can act as effective adjuvants under specific conditions.3−5 Its versatility as a material stems from functional groups that enable facile chemical cross-linking and grafting steps to create hybrid materials.6−13 In the absence of chemical cross-linking agents, the GMS can nevertheless transition from a liquid to a physical gel state in the temperature range 22−37 °C; this change of state can conveniently serve to trigger drug release.6,12,14,15 In contrast, in vivo release of encapsulated agents in cross-linked gelatin typically relies on enzymatic degradation by natural proteases.6,7 GMS can be formed through a variety of methods, including water-in-oil (W/O) emulsions,12,16,17 desolvation,18,19 coacervation,20−22 and spray drying23 though polydisperse populations are inevitable in these approaches. More recently, select studies have employed microfluidics to achieve size uniformity.24−26 To date, microfluidics-based fabrication of GMS has been limited to forming large, cross-linked microspheres (>50 μm in diameter)25 from dilute solutions (≤5 wt % gelatin). In contrast, uncross-linked GMS droplets 35 μm in diameter were fabricated from a 20 wt % gelatin solution in the current work. This approach allows for retention of the temperature-dependent gel-to-liquid phase transition that conveniently aids in its microfluidic generation and in the programmed release of encapsulated oligonucleotides without chemically altering the encapsulated agents. In our prior work, layer-by-layer (LbL) deposition of poly(allylamine) (PAH) and poly(acrylic acid) (PAA) on © 2013 American Chemical Society
gelatin matrixes was found to effectively trap encapsulated DNA at ∼23 °C while promoting DNA release (up to ∼100 nM) at 37 °C.14 When introduced to the surrounding bulk solution, similar concentrations of competitive DNA target strands were shown to induce isothermal “outside-in” disassembly of DNA-linked colloidal satellite assemblies.27 In this work, we take a step toward “inside-out” DNA-mediated disassembly by first encapsulating the competitive DNA target strands inside the template gelatin microparticle of the satellite assembly, then triggering release of the encapsulated DNA from the template at 37 °C to then promote subsequent secondary or competitive hybridization events on satellite particles decorating the surface of the GMS template, as illustrated in Scheme 1. More broadly, incorporating an “intrinsic switching” capability (i.e., eliminating the necessity of a separate step to add the competitive target strands) to modify surface functionality provides new opportunities for developing materials with dynamic surface behavior, such as a camouflage coating that can be programmatically shed to induce subsequent surface activity such as cell adhesion, recruitment, or targeting events.
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EXPERIMENTAL SECTION
Materials. DNA sequences (Integrated DNA Technologies; Coralville, IA), their function, Gibbs free energy of hybridization, ΔGhyb, and estimated duplex melting temperatures, Tm, are shown in Received: September 10, 2012 Revised: March 29, 2013 Published: April 5, 2013 5534
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Scheme 1. Illustrations of (a) Colloidal Satellite Assembly Comprised of a Central, Uncross-Linked Gelatin Microsphere Surrounded by a Layer of Duplex-Functionalized Particles; (b) a Local Segment of the FITC-Labeled Duplexes (Indicated by Green Stars) on the Surfaces of a Small Satellite Particle; and Following Incubation at 37 °C, (c) Competitive Hybridization Partners Are Released from the Central Gelatin Microsphere and Drive Displacement of the Original FITC-Labeled Hybridization Partner Resulting in Longer Duplexes on the Surface of Satellite Particles
Table 1. We use well-established thermodynamic models28−31 and folding servers32,33 to estimate thermodynamic parameters of our
(Devcon, Danvus, MA) and 30-min Permaoxy epoxy (Permatex, Solon, OH). Preparation of DNA-Functionalized Polystyrene Microspheres. Aminated DNA probes (A20) were coupled to 1.1 μm carboxylated polystyrene particles as detailed in a prior report.35 Briefly, 10 μL of microspheres (10 v/w%) were washed and resuspended in 150 μL of a coupling buffer (50 mM MES, 0.05 v% Proclin, pH 5.1). 200 μL of probe DNA (0.1 μM) in TE 7.4 and 25 μL of 1.28 M EDC were then added to the suspension. As detailed in the Supporting Information, SI, a separate study was conducted using polystyrene microspheres functionalized with a higher density of immobilized probe strands. Following a 2 h incubation, suspensions were washed three times in PBS/Tween (0.2% Tween in PBS, pH 7.4). To form primary duplexes on the microsphere surfaces, a 10 μL volume of A20-functionalized microspheres was added to 90 μL of PBS/Tween followed by the addition of 200 μL of either unlabeled or FITC-labeled (indicated with “−F” in sequence nomenclature), primary target in Tris EDTA, pH 8.0 (TE 8.0). After a 30 min incubation at 37 °C, microspheres were washed three times (returning to 37 °C conditions between each wash) to remove thermally dissociated primary targets. Electrophoretic mobility measurements (Zetasizer NS, Malvern Instruments, Worcestershire, UK) were taken and converted to ζ potential values using the Smoluchowski approximation. The ζ potential values (in 10 mM NaCl) were −36.0, −34.6, −43.1, and −40.6 mV for bare, A20-functionalized, A20:P13-functionalized, and A20:P13−F-functionalized polystyrene microspheres, respectively. To optimize conditions promoting subsequent competitive displacement of primary hybridization partners on the satellite particles (adsorbed on GMS template), duplex-functionalized polystyrene microspheres were incubated overnight in a 10 nM or 100 nM solution of P15 or NC secondary targets at 37 °C to mimic DNA release conditions of satellite assemblies. After incubation, the samples were sonicated and prepared for flow cytometry. Microfluidics-Based GMS Fabrication. The capillary microfluidic device was constructed as described in literature36,37 and detailed in the SI to yield a device and gelatin microspheres shown in Figure 1. Secondary DNA target strands (NC or P15) were infiltrated into GMS and a bilayer of PAA/PAH (to prevent premature DNA release prior to 37 °C incubation) was deposited as previously described.14 Briefly, GMS in acetone were centrifuged and resuspended in an aqueous DNA solution (10 μM of P15 or NC sequences) and allowed to incubate for 30 min to drive DNA encapsulation. GMS were then centrifuged and resuspended twice in saline solution (150 mM NaCl) and then in PAH solution (1 mg/mL, pH 7). Following a 10 min incubation time, GMS were centrifuged and resuspended twice in saline solution and then in PAA solution (1 mg/mL, pH 7). Following a 10 min incubation time, GMS were then washed four times in saline solution. Satellite Assembly and DNA Hybridization Activity. For select assembly studies, polydisperse GMS were prepared as previously
Table 1. List of Oligonucleotide Sequences and Their Functiona function immobilized probe perfectly matched primary targets
mismatched primary targets
perfectly matched secondary target noncomplementary target
sequence A20 = 5′-TTT TTT GGA TTG CGG CTG AT-3′ P11 = 3′-AAC GCC GAC TA5′ P13 = 3′-CT AAC GCC GAC TA-5′ M11 = 3′-AAC GCG GAC TA-5′ M13 = 3′-CT AAC GGC GAC TA-5′ P15 = 3′-A CCT AAC GCC GAC TA-5′ NC = 3′-GGA TTG CGG CTG AT-5′
ΔGhyb (kcal/mol)
Tm (°C)
NA
NA
−11.7
63.9
−13.4
65.3
−9.8
57.1
−9.6
53.4
−16.3
69.2
−3.3
30.9
a
Underlined bold text in target sequences indicates an intentional mismatch with the probe sequence. Values for ΔGhyb (Gibbs free energy of hybridization) and Tm (duplex melting temperature) between probe and target species were estimated with Zuker’s MFold server for oligonucleotide solutions using 37 °C, 0.1 mM DNA, and 150 mM NaCl solution conditions.32,33,49 oligonucleotide solutions in which the absolute value of ΔGhyb increases as the hybridization segment increases (e.g., P15 > P13 > P11) and decreases as mismatches are introduced into targets (e.g., P13 > M13). Consistent with suggestions by Markham and Zuker,31 we account for hybridization activity of immobilized oligonucleotides employing the approach of Biancaniello et al,34 which consists in converting an average primary duplex density from flow cytometry measurements (∼200 duplexes/μm2) into an effective molar concentration of immobilized DNA (0.1 mM) in the volumetric shell surrounding each polystyrene microsphere. Span 80, Span 20, Type A gelatin from porcine skin (300 bloom, pI ≈ 8.0), ProClin, 1ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride (EDC), PBS, poly(allylamine hydrochloride) (Mw ≈ 56 kDa), isooctane, octadecyltrimethoxysilane (OTMS), and sodium chloride were purchased from Sigma Aldrich (St, Louis, MO). Poly(acrylic acid, sodium salt) (Mw ≈ 85 kDa) was purchased from Polysciences (Warrington, PA). Mineral oil (viscosity of 34.5−150.0 mm2s−1) and acetone were purchased from VWR international (Arlington Heights, IL). Carboxylated polystyrene microspheres (1.1 μm diameter) were purchased from Bangs Laboratories (Fishers, IN). Ethanol, Tris/ EDTA buffers (pH 7.4 and 8.0) were purchased from Fisher Scientific (Pittsburgh, PA). Two types of epoxy were used, 5-min epoxy 5535
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Figure 1. (a) Photograph and (b) top view schematic (not to scale) of microfluidic device. (c) Micrograph of local gap between the outlet (260 μm inner diameter) and gelatin (12 μm inner diameter) needles used to form un-cross-linked gelatin microspheres. Dashed circles in (b) represent locations of attached needles (90° orientation to the capillary device) where gelatin solution and oil are introduced to the microfluidic device. Flow patterns are indicated in (c) with dashed lines and arrows. Scale bar in (c) represents 100 μm. Phase contrast micrograph (right) showing the uniform gelatin microspheres (GMS) in saline solution (150 mM NaCl) fabricated using microfluidics. Scale bar represents 50 μm. described14 using an emulsion processing approach. Following competitive DNA target encapsulation and deposition of one PAH/ PAA bilayer, GMS were resuspended in 50 μL of saline and added to 100 μL of a polystyrene microsphere population (bare, A20functionalized, A20:P13-functionalized, or A20:P13−F-functionalized) vortexed and incubated for 2 h. After incubation, the suspension was centrifuged at 500 g for 1 min and the supernatant was removed and replaced with PBS/Tween four times to separate out satellite singlets from satellite aggregates. Following four washes, a 20 μL aliquot of supernatant (containing satellite singlets) was prepared for microscopy to separately assess satellite assemblies formed between GMS and one of four polystyrene microsphere populations. To investigate competitive displacement of P13−F primary target strands (on polystyrene satellite microspheres) by P15 secondary or competitive DNA targets (encapsulated inside, then released from monodispere GMS) satellite assemblies were constructed as described above using monodisperse GMS (fabricated via microfluidics) as the microsphere templates and A20:P13−F-functionalized polystyrene microspheres as the satellite particles. Satellite assemblies were incubated overnight at 37 °C in either a microcentrifuge tube (for subsequent flow cytometry studies) or sealed slide (for microscopy studies). Following incubation, suspensions in microcentrifuge tubes were cooled at 4 °C for 10 min to solidify the GMS, sonicated for ∼10 s to drive desorption of satellite particles from template GMS, then centrifuged at 500 g for 1 min. A 100-μL portion of supernatant (consisting of desorbed satellite particles) was removed and prepared for flow cytometry.
Figure 2. Bar graph of primary targets remaining hybridized to A20functionalized polystyrene microspheres following overnight incubation with noncomplementary (NC) and complementary (P15) secondary targets at 37 °C. Each bar represents the average of 2 samples. Duplex values for each sample (consisting of ∼104 particles) are shown as solid dots above and below the average.
strands/μm2 (A20:P13−F) in the presence of noncomplementary NC secondary targets. In the presence of complementary P15 secondary targets, however, the primary duplex density is substantially lowered for each primary target case. This drop in primary duplex density is indicative of competitive displacement of the shorter, weaker hybridization partner by P15. Of the four candidate primary targets, P13−F exhibited the highest thermal stability while still readily undergoing competitive displacement by P15 secondary targets and was thus chosen for subsequent satellite assembly studies. Intriguingly, particles prepared with a higher probe density exhibited little, if any, displacement activity by P15 secondary target strands (see Figure S1 of the SI). Thus, in order to promote displacement activity in the subsequent satellite assembly studies, polystyrene particles were prepared with the lower primary A20:P13−F duplex densities shown in Figure 2. GMS Satellite Assemblies. Microfluidics-based fabrication produced spherical gelatin template particles (35 ± 1 μm in diameter in saline solution) as shown in Figure 1. GMS
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RESULTS AND DISCUSSION Optimizing DNA Hybridization Activity. While our prior work27,35,38−41 investigated primary and competitive hybridization activity at room temperature, the current experimental system requires that primary duplexes resist thermal dissociation, but remain responsive to competitive targets at 37 °C. To identify an appropriate combination of primary and competitive target sequences for these elevated temperature conditions, four primary target sequences (with estimated melting temperatures exceeding 37 °C, as shown in Table 1) were investigated. Polystyrene microspheres with fluorescently labeled primary duplexes were exposed to P15 or NC secondary targets at 37 °C overnight and the surface density of fluorescently labeled duplexes remaining was measured with flow cytometry. As shown in Figure 2, the primary duplex density values range from ∼100 strands/μm2 (A20:M13-F) to a maximum of ∼300 5536
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DNA alone drives adsorption of satellite particles onto GMS template particles. While suspensions of identical particles (i.e., polyelectrolytecoated GMS alone or DNA-functionalized polystyrene microspheres alone) remain dispersed as singlets, attractions between heterogeneous particles (following mixing) drive polystyrene particles to adhere as “satellite” particles on the surface of GMS to form satellite assemblies. Since the primary duplexes are formed prior to mixing of the template and polystyrene particles, adhesion events between the two heterogeneous particle populations must arise from nonspecific attractive interactions such as van der Waals. Given the complex nature of the surface of the gelatin microspheres involving a polyelectrolyte bilayer coating, assessing the distant-dependent Hamaker constant to estimate pairwise van der Waals interactions between DNA-functionalized polystyrenes and the gelatin microspheres, however, is not straightforward for the current experimental system. Although both colloidal species possess a net negative charge, electrostatic repulsion is likely to be weak since prior work14 indicates that the GMS template particles possess only a modest ζ potential value (less than −15 mV at 10 mM NaCl). While it is difficult to identify the nature of these attractive interactions between the negatively charged, polyelectrolyte-coated GMS and DNA-functionalized polystyrene microspheres, net attractions between like-charged species have been attributed in past work42−45 to nonspecific interactions that are not taken into account by classical DLVO theory. Although nonspecific attractive interactions are strong enough to drive the extensive adsorption of polystyrene satellite particles on GMS shown in Figure 3, these attractive interactions are likely modest in magnitude since sonication allowed for satellite particles to desorb from GMS templates for subsequent flow cytometry studies. Next, the impact of heating on the shape and stability of satellite assemblies as well as on the release and activity of encapsulated secondary targets was investigated. In comparing the original satellite assemblies in Figure 3(a) with the same assemblies following overnight incubation at 37 °C in Figure 3(b), heating does not appear to drive desorption of satellite particles or extensive swelling or shape changes of the underlying GMS template. Thus, in the absence of any applied mechanical forces, the assemblies as well as the GMS templates appear stable and mechanically robust under elevated temperature conditions. To then assess if encapsulated DNA (either NC or P15 secondary targets) is released and capable of competitive hybridization activity, heated suspensions were sonicated to drive desorption of satellite particles from assemblies. These desorbed satellite particles were then evaluated via flow cytometry. As shown in Figure 4, the satellite particles desorbed from P15-loaded GMS have a lower density (∼50%) of A20:P13−F primary duplexes than those desorbed from NC-loaded GMS. Prior work demonstrated that brief sonication for 3 s alone does not promote DNA release from PAH/PAA coated GMS.14 Thus, this difference in primary duplex densities for these two cases indicates that P15 is successfully released and capable of competitively displacing P13−F as the hybridization partner. Notably, however, the reduction in the primary duplex density of initially adherent, then desorbed satellite particles shown in Figure 4 was not as dramatic as the case shown in Figure 2 involving dispersed, DNA-functionalized polystyrene particles that were never exposed to GMS particles for satellite assembly formation.
remained dispersed in aqueous solution at room temperature for several days in the absence of chemical cross-linking additives. Figure 3(a),(b) shows resulting satellite assemblies
Figure 3. Phase contrast micrographs of the satellite assemblies comprising a layer of DNA-functionalized microspheres (1.1 μm diameter) adsorbed on GMS at (a) room temperature and (b) following incubation at 37 °C overnight. Scale bars represent 25 μm.
composed of bilayer-coated GMS template surrounded by a single layer of A20:P13−F-functionalized polystyrene particles. Additional micrographs (see Figures S2(a),(b) of the SI) of satellite assemblies are provided in the SI. Given the overall smooth periphery of the satellite assemblies shown in Figure 3 and Figure S2(a) of the SI, the formation of multiple layers of polystyrene particles on one GMS is not apparently favored. This observation is not surprising since suspensions of DNAfunctionalized polystyrene particles (prior to mixing with bilayer-coated GMS) remain as dispersed singlets and are thus unlikely to adhere to one another in the satellite assemblies. Overall, the satellite particle layer appears relatively densely packed in many areas of the gelatin template; however, as shown in additional satellite micrographs in the SI (see Figure S2(a) of the SI) the presence of bare patches between groups of adherent polystyrene particles (see Figure S2(b) of the SI) indicates that a close-packed arrangement is not uniformly achieved in these satellite assemblies. Analogous coverage by satellite particles was also achieved for polydisperse GMS templates incubated with bare, A20-functionalized, A20:P13-functionalized, or A20:P13−F-functionalized polystyrene microspheres (results not shown). The similarities in the satellite structures for the four different polystyrene populations indicate that neither the fluorescent label nor the 5537
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fabrication of uniformly sized gelatin microspheres that served as the template particles and facilitated characterization of the release properties of the resulting temperature-sensitive colloidal satellite assemblies. This system demonstrates the exciting potential to incorporate a programmable, intrinsic “surface switching” capability in materials for applications ranging from injected drug delivery vehicles with sheddable stealth coatings to tissue scaffolds with time-sensitive cell recruiting capabilities.
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ASSOCIATED CONTENT
S Supporting Information *
Device fabrication, competitive hybridization activity, and satellite assemblies. This material is available free of charge via the Internet at http://pubs.acs.org.
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Figure 4. Density of A20:P13−F primary duplexes remaining on polystyrene satellite particles desorbed from satellite assemblies loaded with either complementary P15 or noncomplementary NC secondary targets and incubated overnight at 37 °C.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Author Contributions
This marked difference in the ability to reduce the primary duplex density following exposure to P15 secondary targets for these otherwise identical polystyrene populations indicates that adsorbing polystyrene particles to the GMS hinders subsequent competitive displacement activity on the surface of these satellite particles. In addition to steric issues that hinder primary hybridization activity of immobilized strands,38,46,47 the hindered competitive displacement events on the adherent satellite particles are likely to be further fostered by confined volume conditions restricting the mobility and resulting activity of P15 strands released from the gelatin-based core. Unlike the dispersed suspensions in Figure 2 in which primary A20:P13− F duplexes on polystyrene microspheres are uniformly exposed to the bulk solution of P15 strands, this confined volume condition could limit the accessibility of A20:P13−F duplexes located, for example, at the GMS−polystyrene interface. Despite these restrictions to competitive displacement events, these results in Figure 4 demonstrate that the change in the surface functional groups of a colloidal assemblyhere, the exchange of one duplex for a longer duplex on adsorbed satellite particlesis mediated by secondary targets released from the interior of the assembly itself. This ability to incorporate the release agenthere, the competitive target specieswithin the assembly itself eliminates the need to introduce the release agent separately to the bulk solution. While efforts in the current system focused on incorporating an intrinsic surface switching capability, future efforts lie in tuning the response rate of these colloidal assemblies through choices in both sequence characteristics (e.g., extending base length differences between primary and secondary hybridization partners to drive faster displacement behavior48) and particle characteristics (e.g., adjusting the gelatin concentration to enhance the release rate of encapsulated, secondary target strands for subsequent hybridization activity14).
This manuscript was written through contributions of all authors. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS V.T.M. and J.O.H. gratefully acknowledge funding support of the Georgia Cancer Coalition Distinguished Scholars Program, Army Research Office (W911NF-09-1-0479) and NSF CAREER (DMR-0847436). Partial support was provided to J.O.H. from a GAANN fellowship through Georgia Tech’s Center for Drug Design, Development and Delivery (CD4) and to V.T.M. by a 3M Non-Tenured Faculty Award. A.F.N. gratefully acknowledges support from NSF (CBET-0967298). Flow cytometry was performed at the Institute of Bioengineering and Biosciences (IBB) Core Lab facilities. J.O.H. would like to thank Congwang Ye and Samuele Elisei for instruction and advice on microfludics.
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REFERENCES
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CONCLUSIONS While changes to surface functionalities are typically induced via solute additions to the bulk solution, this study demonstrates the ability to incorporate the solute species itself (here, competitive DNA target strands) inside a central microsphere template and then trigger its release and subsequent activity at 37 °C. Microfluidics enabled the 5538
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