(694e) Injectable, Magnetic “Plum Pudding” Hydrogel Nanocomposites: Improving Externally-Mediated, Enhanced Pulsatile Drug Release

INTRODUCTION:  ?Smart?, polymer-based biomaterials that
respond to specific stimuli have been extensively studied in recent years,
particularly as potential therapeutic release agents.  The temperature-responsive polymer poly(N-isopropylacrylamide) (PNIPAM) has been used to develop
several hydrogels and microgels for drug delivery applications due to the
thermosensitive volume phase transition temperature (VPTT) that this polymer
experiences upon heating.¹  However, such thermosensitive materials alone
are unable to repeatedly release a drug of interest in the body, as safe
application of a thermal stimulus in vivo
is not straight-forward.  Composite
materials that combine thermosensitive materials with nanomaterials that
generate heat in response to external systems (such as gold nanorods,
carbon nanotubes, or superparamagnetic iron oxide nanoparticles (SPIONs)) can overcome
this issue, facilitating an advanced drug releasing mechanism whereby
temperature-dependent swelling/deswelling responses can be non-invasively controlled
in vivo.^2,3

We recently used this approach to produce injectable,
externally-responsive PNIPAM hydrogels in which SPIONs were covalently bound
into the hydrogel network.⁴  An alternating magnetic field (AMF)
was used to heat the SPIONs and induce deswelling of the matrix to produce
pulsatile release of drug over multiple AMF applications.  However, the observed increase in the drug
release was relatively small and too short-lived for practical use (most of the
drug was released after one day).⁴

Building on this result, we have fabricated nanocomposite materials
with SPIONs and thermosensitive microgels entrapped in an injectable
thermosensitive hydrogel matrix, resulting in ?plum pudding?-like nanocomposite
hydrogel materials.  Upon AMF application,
the SPIONs will generate heat that is transferred to the thermosensitive
microgels within the nanocomposite, causing them to deswell and open up free
volume that encourages drug release (Figure 1). 
This proposed release mechanism should also be entirely reversible, so
that when the AMF application is halted, the nanocomposite (and the drug
release rate), will return to its initial state.   This
study not only involves the elucidation of the true mechanism of
externally-induced enhanced drug release, but also the degree to which various
parameters (i.e., the microgel content, the microgel VPTT, the hydrogel
swelling characteristics, etc.) effect the efficacy of enhanced release due to
an AMF exposure over multiple days.

Figure 1: Fabrication of the
injectable ?plum pudding? nanocomposite hydrogels and their proposed AMF-mediated
release mechanism.⁵

EXPERIMENTAL:  Hydrazide-functionalized PNIPAM was prepared
by carbodiimide-mediated conjugation of adipic acid dihydrazide to a
p(NIPAM-AA) copolymer.⁶  Aldehyde-functionalized
dextran was generated by oxidizing dextran with sodium periodate.⁶  The thermosensitive microgels were prepared
via precipitation polymerization of NIPAM (36 mol%), N-isopropylmethacrylamide
(NIPMAM, 58 mol%), and acrylamide (6 mol%) with N,N-methylenebisacrylamide (crosslinker)
and ammonium persulfate (initiator).  SPIONs
were fabricated via coprecipitation and peptized with 8 kDa PEG.⁷ 

The nanocomposites were generated using a double barrel syringe in
which each barrel contains 8 wt% of the hydrogel
precursor polymer (with hydrazide-functionalized PNIPAM and
aldehyde-functionalized dextran in opposite barrels), 8 wt%
thermosensitive microgels, 1 wt% 4 kDa FITC-dextran,
and 5 wt% SPIONs, in 10 mM
PBS.  The hydrazide- and
aldehyde-functionalized polymers rapidly react upon injection to form the
hydrolytically-degradable hydrazone crosslinks of the hydrogel while physically
entrapping the microgels, SPIONs, and drug within the nanocomposite matrix.

Drug release experiments were performed using an AMF setup that allows
for multiple composites (n = 4) to be
held in equivalent positions in the magnetic field while maintaining a constant
baseline temperature of 37°C (or 22°C or 43°C for the varying temperature
experiments).  Drug release experiments
involved immersing the nanocomposites in 4 mL of PBS and applying the AMF for
certain intervals.  For pulsed release
experiments, samples were collected every 10 minutes, including directly before
and after 10 minute AMF pulses (4-6/day) that were applied every 40
minutes.  The FITC-dextran concentration
in each release sample was measured with a fluorescent plate reader.  The increase in the rate of release due to an
AMF application was reported as the percent increase between the measured release
rate following an AMF pulse and the baseline release rate, which was calculated
via linear interpolation between the 2 sample points directly before and the 2
points immediately after the AMF pulse.

RESULTS:  The nanocomposites were confirmed by TGA
to possess ~5 wt% SPIONs and by SQUID analysis to be
superparamagnetic in nature.  Their
degradability was confirmed in accelerated hydrolysis conditions, with full
dissolution of the gel observed after ~240 hours of incubation in pH 1
buffer.  The nanocomposites were quite
elastic, with the storage modulus increasing with microgel content until a
certain critical point (between 12 and 16 wt%
microgel) above which the microgel inhibited the formation of hydrazone
crosslinks upon injection. 

To determine whether or not drug release was taking place via the free
volume mechanism proposed in Figure 1, several different AMF-mediated drug
release experiments were performed.  Drug
release throughout a 2 hour AMF application showed that the composites with
microgel and SPIONs released significantly more drug than composites without
either microgel or SPION content, with the slope of the release versus time
curve roughly double for the AMF-exposed composites.  Following, short 10 minute pulses were
performed that showed that composites with both microgel and SPION content could
achieve much greater enhancements in release due to a pulse compared to
composites without either microgel or SPION content; furthermore, after one day,
only the microgel+SPION composites could achieve
pulsatile AMF-mediated release, with the rate of release after a pulse increasing
as high as fourfold relative to the baseline release. 

Figure 2: Percentage
increases in FITC-dextran release due to an AMF pulse (a) and relative swelling
(b) of the nanocomposites at different baseline temperatures. *p < 0.05 in a pairwise comparison.⁵

Now that the importance of microgels to improving the control over
release over multiple days was confirmed, experiments were performed at
baseline temperatures well below (22°C), slightly below (37°C), and slightly
above (43°C) the VPTT of the microgels (~39°C) to confirm that the phase
transition of the microgels is the origin of the enhanced release (Figure
2).  Figure 2 shows that when the
AMF-controlled system operates from a baseline slightly below the VPTT of the
microgels there is a much greater enhancement of release due to short AMF
pulses, particularly after the first day. 
Overall, these results confirmed our proposed mechanism of AMF-mediated
release.

The influence of various factors on release was then observed.  Studies using microgels with differing PNIPAM:PNIPMAM ratios, and thus different VPTTs, indicated that
microgels with VPTT values slightly above the baseline physiological
temperature facilitated higher pulsatile release, owing to the increased volume
change those microgels can undergo when heated in an AMF.  Composites with higher microgel contents (up
to 10 wt%)
lead to greater external control over release for the entire 5 day period
studied.  In addition, the swelling of
the hydrogel scaffold, and thus the swelling of the nanocomposites, was altered
by replacing a certain percentage of the aldehyde-functionalized dextran with
identically functionalized CMC, a more hydrophilic polymer that enhances the
bulk gel swelling (Figure 3). 

Figure 3: Effect of hydrogel
scaffold swelling on the percent increase in 4 kDa FITC-dextran release due to
a 10 minute AMF application (a) and relative swelling (b) of the
nanocomposites. *p < 0.05 in a
pairwise comparison.

Figure 3 shows that as the amount of CMC relative dextran in the bulk
hydrogel increased, the degree of swelling dramatically increased and the
magnitude and duration of pulsatile drug release both decreased.  This result is attributable to the swelling
bulk hydrogel phase consuming the free volume generated by the deswelling of
the microgels following AMF application.

All of the components of the nanocomposites, and the nanocomposites
themselves, also displayed no significant cytotoxicity via an MTT assay with 3T3 mouse fibroblast cells.  This further suggests that these materials may
be promising candidates as ?smart?, on-demand drug delivery platforms that
could be used to deliver a variety of drugs for therapies that would benefit
from repeated, pulsatile release.

CONCLUSIONS:  The addition of thermosensitive microgels inside injectable, magnetic hydrogel
composite materials significantly improved the externally AMF-controlled
enhanced release.  This enhanced release
occurs due to microgels deswelling during the AMF-mediated heating of the
nanocomposite to create free volume that enhances release.  Such enhancements in pulsatile release can be
tuned by adjusting the microgel VPTT, microgel content, and the hydrogel
swelling characteristics and have shown to persist over multiple days rather
than hours.

REFERENCES:  (1) Pelton, R. Adv. Colloid Interface Sci. 2000, 85, 1-33.  (2) Merino, S. et
al. ACS Nano 2015 [In Press].  (3) Campbell, S.B.; Hoare, T. Curr. Opin. Chem. Eng. 2014,
4, 1-10.  (4) Campbell, S.B.; Patenaude,
M.; Hoare, T. Biomacromolecules
2013, 14, 644-653.  (5) Campbell,
S.; Maitland, D.; Hoare, T. ACS Macro
Lett. 2015, 4, 312-316.  (6) Patenaude, M.; Hoare, T. Biomacromolecules, 2012, 13, 369-378.  (7) Mahmoudi, M. et al. Adv.
Drug Deliv. Rev. 2011, 63, 24-46.

ACKNOWLEDGEMENTS: This research
is funded by the J.P. Bickell
Foundation (Medical Research Grants), and the Natural Sciences and Engineering
Research Council of Canada (Discovery Grant and Vanier Scholarship programs).