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).