(217az) Injectable, Thermoresponsive Hydrogels With Tunable Cross-Linking Kinetics Based On Ketone and Aldehyde-Functionalized Pre-Polymers

Injectable,
thermoresponsive hydrogels with tunable cross-linking kinetics based on ketone
and aldehyde-functionalized pre-polymers

Mathew Patenaude
and Todd Hoare

Department of
Chemical Engineering, McMaster University, Hamilton, Ontario, L8S 4L7

E-mail:  patenamj@mcmaster.ca 
|  hoaretr@mcmaster.ca

Introduction

          Hydrogels  have been widely investigated
for use in biomedical applications due to their physicochemical  and 
mechanical  similarity  to  the  mammalian  extracellular matrix. Hydrogels 
based  on  poly(N-isopropylacrylamide)  (pNIPAM)  are particularly  attractive 
for potential  in  vivo  application  as ?smart? hydrogels because  they
possess  a  volume  phase  transition  temperature  (VPTT)  close  to
physiological  temperature.  Below the VPTT, the polymer network is swollen; exceeding 
this  VPTT  leads  to  a  collapse of  the  hydrogel  network  and  subsequent 
expulsion  of  water, potentially  facilitating  entrapment and/or pulsatile
release of  therapeutics  in  drug delivery applications as well as tunable
cell adhesion/release in tissue engineering applications.

Injectable, in
situ-gelling hydrogels hold particular interest for in vivo applications
given that they can easily be administered into the body without the need for
surgical implantation.  We  have  previously  demonstrated  the  fabrication 
of the first injectable, degradable, and covalently-crosslinked pNIPAM 
hydrogels composed  of  hydrazide-functionalized  pNIPAM  and  aldehyde
functionalized  pNIPAM  precursor  chains.¹ Following 
their  co-extrusion through  a  double  barrel  syringe,  these  precursors 
can  form  a  covalently  cross-linked  hydrogel  network  via  formation  of  hydrolysable
hydrazone bonds  between  the  precursor  chains; by synthesizing the precursor
polymers below the molecular weight of the renal glomerular membrane exclusion
limit (i.e. below 40 kDa), the hydrogel can degrade over time and clear from
the body.²  The  ability  of  these  gels  to 
rapidly form  in  situ  following  the  co-extrusion  of  complimentary 
precursor copolymers  makes  these  systems  attractive  candidates  as 
injectable biomaterials.  However, the extremely rapid rate of gelation results
in gel formation within seconds following extrusion, providing minimal time for
self-diffusion of the mixed precursor chains within the pre-gel mixture and
leading to hydrogels with nanoscale domains of un-crosslinked (unmixed)
precursor solutions and/or highly heterogeneous gel structures.  Furthermore,
while gelation time can be slowed by reducing the functional group content
and/or the functional polymer content to enable e.g. spreading of the pre-gel
mixture on a surface or self-diffusion-driven mixing, this approach also
results in a weaker hydrogel following gelation. As such, it is of interest to
reduce the rate of gelation without significantly affecting the mechanical or
physicochemical properties (in particular, the cross-link density) of the final
hydrogel. 

To this end, we
have designed a synthetic strategy to control the rate of hydrazone hydrogel
network formation by cross-linking hydrazide-functionalized pNIPAM with pNIPAM
copolymers possessing a mixture of aldehydes (fast reacting) and ketones (slow
reacting). In this way, we can generate injectable hydrogels with a range of
formation and degradation kinetics as well as tunable internal morphologies.

Materials and Methods

          Protected
aldehyde and ketone monomers were synthesized according to Figures 1 and 2,
respectively. Precursor PNIPAM based copolymers were fabricated according to Figure
3, with hydrogels made from hydrazide-functionalized PNIPAM (nucleophilic
precursor) and PNIPAM consisting of a range of aldehyde to ketone functionality
(electrophilic precursor). The molecular weight of precursor PNIPAM copolymers
was determined using gel permeation chromatography. The degree of hydrazide
functionality in the hydrazide functionalized PNIPAM precursor chains was
determined using conductometric titration. Aldehyde and ketone functionality
were determined using proton NMR calibrated with a tetramethyl silane standard.

          Nucleophilic and electrophilic
precursors were mixed at constant polymer weight percent via co-injection
through a double barrel syringe, as shown in Figure 3. The rate of gel
formation for gels made from a range of ketone to aldehyde content was
determined using infrared spectroscopy by monitoring the rate of hydrazone peak
appearance around 1614-1602 cm^-1 and 3344-3263 cm^-1.
Reversible thermoresponsiveness was determined gravimetrically by incubating
the gels at alternating temperatures of 25 °C and 37 °C. The degradability of
these gels was demonstrated by incubating them in different concentrations of
hydrochloric acid and determining the percent of gel loss over a period of time
gravimetrically. Gel heterogeneity was assessed using light transmission
measurements and X-ray scattering.    

Results and Discussion

          The rate of gel network formation
was determined using Fourier transform infrared spectroscopy operating in
reflectance mode. A significant decrease in gel cross-linking kinetics was
observed as the fraction of the precursor ketone content was increased (Figure
4). Using this method, we have determined that complete gel cross-linking
occurs in around 25 hours for gels composed of electrophilic chains possessing
100% ketone content and around 2 hours for gels possessing 100% aldehyde
content, with functional gels passing the vial inversion test produced within
~3 minutes for aldehyde-only gels and ~30 minutes for ketone-only gels.
Mixtures composed of electrophilic copolymer chains consisting of a 50/50 mix
of aldehydes to ketones formed fully cross-linked solid gels at an intermediate
time of around 5 hours, with functional gels being produced after ~10 minutes. 
Transmission studies conducted at 500 nm demonstrated that the degree of
homogeneity increases as the precursor ketone content is increased and the cross-linking
kinetics are slowed (Figure 5). X-ray scattering and atomic force microscopy
results probing the homogeneity of the ketone versus aldehyde-functionalized
hydrogels will be presented.   

Conclusions

          The
rate of gel formation in hydrazone-crosslinked networks can be tuned by
changing the ratio of aldehyde to ketone functional groups in the electrophilic
precursor polymer mixture.  Gelation times ranging from 3 to 30 minutes can be
achieved, with slower cross-linked hydrogels exhibiting improved homogeneity
without sacrificing mechanical strength.  This approach can be used to make
injectable hydrogels adaptable to a much wider range of potential biomedical
applications (i.e. cell encapsulation, thin film formation, etc.).

Acknowledgements:  Funding from the Natural Sciences Engineering Research Council of
Canada (NSERC) is gratefully acknowledged

References

(1)     Patenaude, M.; Hoare, T. ACS Macro Letters
2012, 1, 409?413.

(2)     Patenaude, M.; Hoare, T. Biomacromolecules
2012, 13, 369?78.