The hydrogel template method for fabrication of homogeneous nano/microparticles.

Abstract

Nano/microparticles have been used widely in drug delivery applications. The majority of the particles are prepared by the conventional emulsion methods, which tend to result in particles with heterogeneous size distribution with sub-optimal drug loading and release properties. Recently, microfabrication methods have been used to make nano/microparticles with a monodisperse size distribution. The existing methods utilize solid templates for making particles, and the collection of individual particles after preparation has not been easy. The new hydrogel template approach was developed to make the particle preparation process simple and fast. The hydrogel template approach is based on the unique properties of physical gels that can undergo sol-gel phase transition upon changes in environmental conditions. The phase reversible hydrogels, however, are in general mechanically too weak to be treated as a solid material. It was unexpectedly found that gelatin hydrogels could be made to possess various properties necessary for microfabrication of nano/microparticles in large quantities. The size of the particles can be adjusted from 200 nm to >50 microm, providing flexibility in controlling the size in drug delivery formulations. The simplicity in processing makes the hydrogel template method useful for scale-up manufacturing of particles. The drug loading capacity is 50% or higher, and yet the initial burst release is minimal. The hydrogel template approach presents a new strategy of preparing nano/microparticles of predefined size and shape with homogeneous size distribution for drug delivery applications.

Full text

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The hydrogel template method for fabrication of homogeneous nano/microparticles
Ghanashyam Acharyaa, Crystal S. Shinb, Matthew McDermottb, Himanshu Mishraa, Haesun Parka,
Ick Chan Kwonc, Kinam Parka,b,⁎
aDepartments of Biomedical Engineering and Pharmaceutics, Purdue University, West Lafayette, IN 47907, USA
bAkina, Inc. 1291 Cumberland Ave., West Lafayette, IN 47906, USA
cBiomedical Research Center, Korea Institute of Science and Technology, Cheongryang, Seoul, 130-650, Republic of Korea
a b s t r a c t a r t i c l ei n f o
Article history:
Received 21 July 2009
Accepted 28 September 2009
Available online xxxx
Keywords:
Hydrogel template
Microfabrication
Microparticle
Homogeneous size
Drug delivery
Nano/microparticles have been used widely in drug delivery applications. The majority of the particles are
prepared by the conventional emulsion methods, which tend to result in particles with heterogeneous size
distribution with sub-optimal drug loading and release properties. Recently, microfabrication methods have
been used to make nano/microparticles with a monodisperse size distribution. The existing methods utilize
solid templates for making particles, and the collection of individual particles after preparation has not been
easy. The new hydrogel template approach was developed to make the particle preparation process simple
and fast.
The hydrogel template approach is based on the unique properties of physical gels that can undergo sol–gel
phase transition upon changes in environmental conditions. The phase reversible hydrogels, however, are in
general mechanically too weak to be treated as a solid material. It was unexpectedly found that gelatin
hydrogels could be made to possess various properties necessary for microfabrication of nano/microparticles
in large quantities. The size of the particles can be adjusted from 200 nm to >50 µm, providing flexibility in
controlling the size in drug delivery formulations. The simplicity in processing makes the hydrogel template
method useful for scale-up manufacturing of particles. The drug loading capacity is 50% or higher, and yet the
initial burst release is minimal. The hydrogel template approach presents a new strategy of preparing nano/
microparticles of predefined size and shape with homogeneous size distribution for drug delivery
applications.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
Nano/microparticles play an important role in drug delivery. They
have been usually prepared by well known emulsion methods [1,2].
Nano/microparticles prepared by the conventional emulsion methods
have polydisperse size distributions. The shape of the particles is
limited to sphere, and thus, it has been difficult to examine the effect
of size and shape on biological responses [3]. These particles also
possess sub-optimal drug loading and release properties. Their drug
loading capacity is usually less than 10% with an average of about 5%,
while the drug loading efficiency is less than 50% [4]. It is important to
develop nano/microparticulate formulations with higher drug loading
efficiency and capacity.
Microfabrication methods, because of their ability to control
microarchitecture and feature size, have been used successfully to
develop novel nano/microparticles for applications in drug delivery.
Optically encoded microparticles have been prepared by in situ
photopolymerization in microfluidic channels [5]. Mesoporous silicon
particles have been used as multistage drug delivery systems [6].
Several soft lithography methods, such as microcontact hot printing
[7], particle replication in nonwetting templates [8], and step and
flash imprint lithography [9], have been developed using polymer
templates to prepare homogeneous particles. These methods, despite
their success in producing homogeneous microstructures, are not
readily applicable for fabrication of particles designed for drug
delivery or for scale-up. The presence of any impurities resulting
from in situ polymerization steps will prevent clinical applications,
and the difficulty in mass production resulting from multi-step
particle recovery procedures could make commercial applications
difficult. Successful translation of microfabrication technique to
development of clinically useful drug delivery formulations requires
development of easier and faster processing steps that do not affect
the desirable drug loading and release properties.
2. The hydrogel template approach
Since the drug-containing nano/microparticles are prepared for
ultimate use in clinical applications, it is necessary to produce
particles in large quantities in a reproducible manner by simple
Journal of Controlled Release xxx (2009) xxx–xxx
⁎ Corresponding author. Purdue University, Weldon School of Biomedical Engineer-
ing, 206 S. Martin Jischke Drive, West Lafayette, IN 47907-2032, USA. Tel.: +1 765 494
7759; fax: +1 765 496 1912.
E-mail address: kpark@purdue.edu (K. Park).
COREL-05244; No of Pages 6
0168-3659/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2009.09.032
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microfabrication processing steps. Thus, it is desirable to develop a
method that can collect formed particles by simply dissolving the
templates in aqueous solutions. It would be even more desirable if the
drug-containing particles can be delivered while they are still in the
templates. For this reason, a hydrogel system that can be fully
dissolved in aqueous solution was contemplated. It was not clear,
however, whether this approach would be feasible, because it is well
known that hydrogels, especially sol–gel phase reversible hydrogels,
do not have a mechanical strength high enough to be used as
templates, and the condition for sol–gel phase transition can be too
harsh for loaded drugs. It was unexpectedly found, however, that
gelatin could be manipulated to possess a mechanical strength
sufficiently high enough for use as templates with a gel-to-sol
transition temperature low enough not to adversely affect the loaded
drugs.
Fig. 1 shows the main idea of the hydrogel template approach. The
first step is to form a pattern of vertical posts on a silicon wafer master
template (Fig. 1A). If an intermediary silicone rubber template is to be
used, then the vertical posts on the master template will become
vertical cavities. On top of the master template is poured a warm
aqueousgelatin solution,and then the temperatureis lowered to form
a gelatin hydrogel imprint(Fig. 1B).Oncethe gelatinlayer is solidified,
the gelatin mold is peeled off and the gelatin mold is placed on the flat
surface to expose thecavities (Fig. 1C).The cavities in the gelatin mold
are filled with a solution or a paste of drug/polymer mixture (Fig. 1D).
In this study, poly(lactic-co-glycolic acid) (PLGA) was used as a model
biodegradable polymer, and Nile Red, a fluorescent probe, was used
for easy visualization unless specified otherwise. The organic solvent
present inside the cavities is removed by drying, and the formed
particles are collected by simply dissolving the hydrogel mold,
followed by centrifugation or filtration (Fig. 1E).
3. Materials and methods
3.1. Materials
Gelatin (from porcine skin, Type A, 300 bloom), 1,6-diphenyl-
1,3,5-hexatriene (DPH), 6-propionyl-2-dimethylaminonaphthalene
(Prodan), and Nile Red were purchased from Sigma (St. Louis, MO,
USA), and poly(lactic-co-glycolic acid) (PLGA) of different molecular
weights (MW 36,000, IV 0.7 dL/g; MW 65,000, IV 0.82 dL/g; MW
112,000, IV 1.3 dL/g) were purchased from Lactel (Pelham, AL).
3.2. Fabrication of a silicon wafer master template by photolithography
A silicon wafer was spin coated with SU8 2010 photoresist
(Microchem, MA) at 3500 rpm for 30 s followed by baking at 95 °C
for 3 min. The photoresist coated silicon wafer was exposed to UV
radiation through a mask containing a 10 µm diameter circular
pattern for 12 s. Please note that the diameter can be changed to any
other value of interest, and any specific sizes used in this article are
just for a demonstration purpose. After exposure, the silicon wafer
was post baked at 95 °C for 3 min followed by development in SU-
8 developer for 2 min. The silicon wafer was rinsed with isopropanol
and dried with nitrogen gas. The wafer thus fabricated contained
10 µm diameter wells.
3.3. Fabrication of silicon master templates by electron beam (e-beam)
lithography
Making surface patterns of submicron sizes requires e-beam
lithography. Circular patterns of 500 nm squares were designed using
theAutoCAD2007program.A3-in.diametersiliconwafercoveredwith
1 µm thick SiO2layer (University Wafer, South Boston, MA) was spin
coated with poly(methyl methacrylate) (PMMA, Microchem, Newton,
MA) photoresist of 300 nm thick layer using a spin coated at 3500 rpm
for 30 s (SCS P6708 spin coating system, Indianapolis, IN). The coated
PMMA photoresist layer was exposed to e-beam in a preprogrammed
pattern using a Leica VB6 High Resolution Ultrawide field e-beam
lithography Instrument (Bannockburn, IL) operating at 100 KV,
transmission rate 25 MHz current 5 nA. After e-beam lithography, the
silicon wafer was developed in 3:1 isopropanol:methyl isobutyl ketone
solution to remove exposed regions of the photoresist. A 5 nm thick
chromium layer and a 20 nm thick gold layer were successively
deposited on to this pattern followed by liftoff of the residual PMMA
film in refluxing acetone. The pattern was transferred to the underlying
silicon oxide by deep reactive ion etching with SF6/O2plasma. The
generated silicon master template was used in the fabrication of
hydrogel templates.
3.4. Fabrication of hydrogel templates
A clear gelatin solution (30% w/v in aqueous solution, 10 ml) at
50–55 °C was transferred with a pipette onto a silicon master
template (3in. diameter) containing circular pillars (e.g., of 10 μm
diameterand10 μm height).The gelatinsolution wasevenlyspreadto
form a thin film completely covering the master template and cooled
to 4 °C for 5 min by keeping it in a refrigerator. Cooling resulted in
formation of a gelatin template which was subsequently peeled away
from the master template. The obtained gelatin template was ∼3in. in
diameter, and contained circular wells (e.g., of 10 μm diameter and
10 μm depth). The gelatin template was examined under a bright field
reflectance microscope to determine its structural integrity. The
preliminary experiment suggested that a solution of 30% gelatin
resulted in templates which were elastic and mechanically strong
enough for further processing.
3.5. Polymeric microstructures
200 μlof40%PLGAsolution(MW112,000,IV1.3 dL/g)w/vinCH2Cl2
doped with Nile Red was transferred with a pipette onto a gelatin
templatecontainingcirculartrenchesof,e.g.,10 μmdiameteranddepth.
The PLGA solution was evenly spread. The PLGA-filled gelatin template
was left to dry at room temperature (∼25 °C) for 5–10 min. The gelatin
templatefilledwithPLGAsolutionwascharacterizedbybrightfieldand
fluorescence microscopy.
3.6. Collection of free microstructures
Gelatin templates filled with PLGA solution were left at room
temperature for 10 min to remove most of CH2Cl2solvent from the
templates. For complete removal of the solvent, extended drying at
elevatedtemperaturesorfreezedryingcanbeused.Abatchof10gelatin
templates were dissolved in a 100 ml beaker containing 50 ml of
Nanopure water at 40 °C and gently shaken for 2 min to completely
dissolve thetemplates.This step resulted in completerelease of thefree
and isolated microstructures into the solution. The dispersion was
transferred into conical tubes (15 ml) and centrifuged for 5 min
(Eppendorf Centrifuge 5804, Rotor A-4-44, at 5000 rpm, 19.1 RCF,
(relativecentrifugalforce).Thepelletobtainedupon centrifugation was
Fig. 1. Schematic description of the hydrogel template approach for fabrication of
homogeneous microstructures. See text for descriptions of steps A–E.
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freezedriedandstoredinarefrigerator.Thispelletuponresuspensionin
1 ml of Nanopure water formed free and isolated particle dispersion.
3.7. Characterization of polymer microstructures
The polymer microstructures were visualized by bright field,
confocal fluorescence imaging and scanning electron microscopy
(SEM). Bright field and confocal fluorescence imaging was performed
on an OlympusSpinningDisc ConfocalImaging Microscope BX61-DSU
(Center Valley, PA) which is equipped with Intelligent Imaging
Innovations Slide Book 4.0 software for automated Z-stack and 3-D
image analysis. Scanning electron microscopy was performed on FEI
NOVA nano SEM (Hillsboro, OR) and Hitachi 4800 SEM (Pleasanton,
CA).
4. Results
In a typical experiment, a silicon wafer master template having
vertical posts of 10 μm diameter and height was fabricated by
photolithography. The prepared gelatin template contained the
exact imprint of the features present on the silicon wafer (Fig. 2A).
Hydrogel templates could also be prepared with other hydrogel-
forming materials, such as alginate, agarose, and carrageenan. These
materials form hydrogel templates under different conditions, and
they also have different mechanical properties. As seen in Fig. 2B,
cavities in the gelatin template were readily filled with a Nile Red-
containing PLGA solution without forming a scum layer. The formed
microstructures can be obtained by simply dissolving the gelatin
template in aqueous solution. In the example in Fig. 2, however, the
PLGA-filled gelatin template was pressed on a glass slide to transfer
the PLGA microstructures on to it so that they could be imaged by
SEM. The SEM image in Fig. 2C revealed that the microstructures were
solid cylinders of 10 μm diameter and 10 μm in height, corresponding
to the cavity dimension in the gelatin template. The tilted side views
of the microparticles are clearly visible in Fig. 2C. Free and
homogeneous polymer particles were recovered from gelatin tem-
plates by immersing them in warm water (40 °C) for 2 min. Warm
water completely dissolved the gelatin template and released the free
polymer microstructure into the aqueous solution (Fig. 2D). The
solution containing PLGA particles was centrifuged and freeze dried to
obtain free PLGA particles.
Whilethehydrogel templateapproachenabledfacile fabrication of
homogeneous microparticles, it was not apparent whether the same
approach could be used to fabricate submicron size particles. The
main question was whether the hydrogel template could hold the
dimension of cavities at the submicron level, and whether the cavities
could be filled with PLGA solution or paste. Fig. 3 shows that the same
procedures used to make larger size particles (e.g., 10 µm particles in
Fig. 2) could be easily applied to fabricate particles with diameter as
small as 200 nm. This demonstrates that the hydrogel template
approach can be applied for production of submicron structures of
predefined dimensions.
As an example to demonstrate how easily this method works to
make diverse structures and sizes, a gelatin template having cavities
of a computer keyboard design was fabricated. A computer keyboard
containing letters, numerals, and symbols presents rather complicat-
ed geometries. As shown in Fig. 4, gelatin templates can be used to
imprint not only simple characters (such as I, O, C, etc,) but also
complex geometries (such as &, $, #, π, etc.), thus demonstrating its
precision imprinting capability. The SEM image of the gelatin
template (Fig. 4A) clearly shows that the hydrogel template strategy
possesses a high precision imprinting capability. The space between
each key is clearly visible and even the smallest spaces inside the
Fig. 2. Fabrication of homogeneous 10 µm PLGA microstructures using the hydrogel
templatemethod:(A)brightfieldimageofagelatinhydrogeltemplate;(B)fluorescence
image of a hydrogel template filled with PLGA solution containing Nile Red; (C) SEM
image of free standing PLGA microstructures obtained by pressing the gelatin template
on a glass slide; (D) fluorescence image of free PLGA microstructures obtained after
dissolving gelatin templates. (Scale bars correspond to 10 µm).
Fig. 3. Fabrication of submicron PLGA particles by the hydrogel template approach. PLGA
nanoparticles with diameters of 500 nm (A–D) and 200 nm (E–H). SEM images of gelatin
templates (A and E); fluorescence image of a gelatin template filled with PLGA-Nile Red
solution(BandF);SEMimagesoffreePLGAparticles(CandG);andfluorescenceimagesof
free PLGA particles (D and H).
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letters retained their integrity. The line thickness of each character is
5 µm. The filling of the keyboard template with PLGA-Nile Red
solution was very clean and no formation of scum layer was observed
(Fig. 4B). The gelatin template can also be used to form the letters of
the key board in the projection mode (Fig. 4C). The high precision
imprinting capability was maintained. Fig. 4D shows the letters
obtained by dissolving the gelatin template in Fig. 4B in water. For
applications in drug delivery, the shape of delivery systems may need
to be more compact for increasing the reservoir space, and thus,
particles of different shapes were prepared, such as circles, diamonds,
triangles, squares, stars, donuts, packmans, and crosses (Fig. 5).
For particulate systems used in drug delivery, a high drug loading
capacity and control of the drug release profile are important. As an
example, PLGA particles containing felodipine were prepared using
the hydrogel template method. The prepared PLGA–felodipine
microparticles were examined for their drug loading efficiency and
drug release kinetics in vitro. The analysis confirmed that the
felodipine-loaded microparticles contained as high as 50% of the
drug with an encapsulation efficiency of 80%. The remaining 20% of
the drug was recovered and reused and thus, the actual loss is
minimal. Despite such a high drug loading the initial burst release of
felodipine was about 10% (Fig. 6). Other microparticles containing
different drugs showed different release profiles, but all of them
exhibited very low initial burst release profiles. More studies will be
necessary to maximize the usefulness of this approach, but the initial
drug loading study shows that the drug can be loaded at concentra-
tions much higher than possible by conventional emulsion method.
5. Discussion
Phase reversible hydrogels have been used in tissue engineering,
drug delivery, diagnostics, and as biosensors [10–13]. Hydrogels, to
the best of our knowledge, have never been used as dissolvable
template materials in the preparation of nano/microparticles. It was
commonly thought that the hydrogel templates would be too weak
for the intended applications. Indeed, most synthetic physical
hydrogels are mechanically very weak, limiting their usefulness as
templates for microfabrication. To demonstrate the proof of principle
of the strategy, we examined various natural hydrogels, and selected
gelatin as a model system for preparation of hydrogel templates
because it was unexpectedly found to have many properties ideal for
our applications. Gelatin undergoes a sol–gel phase transition at
40∼45 °C. Because gelatin hydrogels turned out to be highly elastic
and mechanically robust at the concentration used in our study, it can
withstand the physical manipulation during the template preparation
and filling of cavities with drug/polymer mixture. The gelatin
template maintained a drug–polymer mixture inside the cavities
and prevented diffusion of the drug or polymer into the template.
There are several advantages of the hydrogel template approach. It
does not require in situ thermal or photopolymerization procedures
that require purification of the formed nano/micro structures.
Hydrogel templates can be readily prepared from a microfabricated
siliconwafermastertemplate, orfromanintermediate siliconerubber
template, by simply covering the master template followed by
cooling. The hydrogel template strategy is broadly applicable, mild,
and readily scalable for production of homogeneous nano/micro-
structures in large quantities. It also offers precise control over
structural geometries and lateral dimensions in the range of 200 nm
to 50 μm and larger.
Currently available drug delivery systems prepared by emulsion
methods have rather low drug loading capacity, usually much less
than 10% of the total weight, and often show significant initial burst
release of the drug, which can be a half or more of the total loaded
drug [14]. Monodisperse particles prepared by microfluidic emulsi-
fication also showed such high initial burst release [15]. The drug
loading into nano/microparticles prepared by the hydrogel template
method was much higher than that achieved by conventional
methods, most likely due to the fact that the drug/polymer particles
inside the cavities encounter almost no water until they are hardened
by drying. In the emulsion methods, the drug/polymer particles are in
constant contact with a large amount of water from the emulsion
state, probably leading to substantial loss of the drug, as well as
accumulation of the drug on the particle surface.
Fabrication of homogeneous particles of non-spherical geometries
is important in understanding the influence of shape of the nano/
micro structureson their biological behavior, such as blood circulation
times, targeting, and cellular internalization for intravenous admin-
istration of submicron sizes [16,17]. But the effect of the shape on
biological response has not been fully examined, mainly due to the
Fig. 4. A gelatin template imprinted with a computer keyboard demonstrating the
precision imprinting capability. (A) SEM image of a partiallydried gelatin template with
keyboard imprint as trenches; (B) fluorescence micrograph of a gelatin template filled
with PLGA–Nile Red solution; (C) SEM image of a partially dried gelatin template with
keyboard imprint as projections; (D) fluorescence micrograph of free letters obtained
by dissolving the gelatin template in water.
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lack of availability of such particulate systems. Traditional emulsion
methods of preparing nano/microparticles result in spherical shape,
and only recently, formation of particles in a variety of different
geometries became possible as a result of advances in nano/
microfabrication techniques. Microstructures with complex geome-
tries influence the anisotropic interactions with biomolecules through
steric or spatially segregated surface functionalities. Phagocytosis by
macrophages and the renal and hepatic clearance of microstructures
are also known to be influenced by the geometry of nano/
microstructures [18].
It will be useful to generate a comprehensive library of micro-
structures with complex geometries to examine their potential
benefits of targeted drug delivery. Figs. 2 and 3 show formation of
disc shape particles, and Figs. 4 and 5 show the capability of making
microstructures with almost any geometry. Furthermore, the hydro-
gel template approach can also be used to prepare multi(n)-layered
microstructures by simply filling the cavities of the template n times.
Thus, the same drug can be loaded at different concentrations or
different drugs can be loaded into the same particles. Such multi-
layered structures are useful in making even more advanced
structures, e.g., capsules, for delivery of fragile drugs, such as peptide
and protein drugs, and in making multi-functional systems. The ease
and flexibility of the hydrogel template approach are expected to find
applications in delivery of diverse drugs, ranging from low molecular
weight hydrophobic drugs to high molecular weight proteins. The
hydrogel template approach provides a new option of fabricating
microstructures with various geometries and with multi-functional
systems.
6. Conclusions
A hydrogel template approach was developed to prepare homo-
geneous nano/micro structures of various geometries. This can be
readily applicable for the large scale production of nano/microparti-
cles because of its easiness in producing particles. The flexibility in
forming different microstructures allows the approach to incorporate
diverse drugs with various hydrophilic characters and molecular
weights. The study demonstrated production of particles in various
geometries and sizes with high precision. Drug loading is also shown to
be very high with controllable drug release kinetics. The hydrogel
template approach provides a new avenue of preparing nano/micro-
particles for drug delivery.
Acknowledgment
This project was supported in part by the Showalter Trust Fund
from Purdue Research Foundation and by the Global Research
Laboratory Project of Ministry of Education, Science and Technology
of South Korea.
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