Name: an injectable and drug-loaded supramolecular hydrogel for local catheter injection into the pig heart
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This work was funded by the Ministry of Education, Culture and Science (Gravity program 024.001.035), the Netherlands Organisation for Scientific Research (NWO), the European Research Council (FP7/2007-2013) ERC Grant Agreement 308045 and conducted within the LSH TKI framework. This research forms part of the Project P1.03 PENT of the research program of the BioMedical Materials institute, co-funded by the Dutch Ministry of Economic Affairs. This project was supported by ICIN - Netherlands Heart Institute (<a href="www.icin.nl">www.icin.nl</a>) and the &#8220;Wijnand M. Pom Stichting&#8221;. The authors would like to thank Henk Janssen and Joris Peters for the synthesis of the UPy-hydrogelator and Remco Arts for providing the mRuby2. We thank Bert Meijer, Tonny Bosman, Roxanne Kieltyka, Stijn Kramer, Joost Sluijter, Imo Hoefer, and Frebus van Slochteren for the many useful discussions and Marlijn Jansen, Joyce Visser, Grace Croft and Martijn van Nieuwburg for technical assistance.

NOTE: All in vivo experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals by the Institute of Laboratory Animal Resources. Experiments were approved by the Animal Experimentation Committee of the Medicine Faculty of the Utrecht University, the Netherlands.
1. Formulation of the Hydrogel
<ol>
	<li>To prepare 1 ml of the 10 wt% gel, dissolve 100 mg of the UPy-hydrogelator in a vial in 900 &#956;l PBS pH 11.7 by stirring at 70 &#176;C for 1 hr using a magnetic stirrer. Afterwards cool the viscous solution down to room temperature. The solution should now have a pH of approximately 9.0. This solution can be stored for several days.</li>
	<li>Pipette the appropriate amount of drug or biomolecule that is dissolved in neutral PBS into the viscous solution and stir for 10 min to reach a uniform distribution. If the solution becomes too viscous, shortly warm it with hot water.</li>
	<li>Place the solution for 1 hr under an UV-lamp to sterilize.</li>
</ol>
2. Analysis of Hydrogel
<ol>
	<li>Rheological assessment of the solution
	<ol>
		<li>Before loading the gel, mount the 25 mm plate-plate geometry in the rheometer, set the temperature to 20 &#176;C and load the plate with water to prevent evaporation of the gel during measurement.</li>
		<li>Pipette 300 &#956;l of the solution onto a 25 mm plate-plate geometry on a rheometer maintained at 20 &#176;C and lower the plates to obtain a 0.5 mm gap distance.</li>
		<li>Record shear viscosity as function of shear stress from 0.1 to 500 Pa with 10 points per decade.</li>
	</ol>
	</li>
	<li>Rheological assessment of the gel
	<ol>
		<li>Pipette 300 &#956;l of the solution onto the plate and pipette a total of 4.2 &#956;l of 1 M HCl at different places on the solution to induce the gel formation.</li>
		<li>Lower the plates to a gap distance of 0.5 mm and let the gel cure for approximately 30 min. During this curing process, measure the storage and loss moduli at low frequency and strain, for example respectively 1 rad/sec and 0.5%.</li>
		<li>After the gel has cured (after approximately 30 min), record storage and loss moduli as function of the frequency (0.1-100 rad/sec) and subsequently as function of the strain (0.1-1,000%).</li>
	</ol>
	</li>
</ol>
3. Erosion and Release Experiments
<ol>
	<li>Transfer 100 &#181;l of the viscous solution containing the drug or biomolecule into a poly(ethylene terephthalate) hanging cell culture insert for 24-wells plate with pore size 8.0 &#181;m. To prevent leakage of the polymer solution while in the liquid phase, cover the bottom of the inserts with Parafilm (Figure 2A).</li>
	<li>Immediately afterwards pipette 1.4 &#181;l of 1 M HCl on top of the viscous solution to reduce the pH to approximately 7.0-7.2 and let the gel cure inside the insert for about 30 min.</li>
	<li>Remove the Parafilm from the inserts, place the insert in a 24-well plate and fill the well with 800 &#181;l PBS pH 7.4. Incubate the plate at 37 &#176;C with slow rocking or shaking movement. To prevent evaporation of the solvent, fill remaining empty wells with PBS and seal the 24-well plate with Parafilm (Figure 2B).</li>
	<li>Periodically refresh the PBS and analyze the removed PBS for released UPy erosion product or drug/biomolecule.
	<ol>
		<li>Quantify UPy erosion products or pirfenidone by measuring UV absorbance at 265 nm or 320 nm respectively. For fluorescent protein mRuby2 measure fluorescence emission at 587 nm after excitation at 559 nm.</li>
		<li>Translate measured absorption/emission values to concentrations via predetermined calibration curves.
		<ol>
			<li>Prepare calibration curves dissolving a series of known concentrations of the analyte in buffer and measure the UV absorbance or fluorescent emission of these samples. Interpolate the data using a linear function to determine the concentration of the unknown samples. For non-fluorescent proteins use ELISA detection6.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Local Injection via a Catheter
<ol>
	<li>Induction of myocardial infarction
	<ol>
		<li>After 12 hr of fasting, excluding water, sedate the pig in its stable by injecting midazolam 0.4 mg/kg, ketamine 10 mg/kg and atropine 0.014 mg/kg intramuscularly.</li>
		<li>Administrate sodium thiopental 5 mg/kg intravenously to induce anesthesia and intubate the pig with an endotracheal tube. Perform balloon-ventilation at a rate of 12/min if needed while transporting the animal to the operating theater.</li>
		<li>On arrival at the operation theater immediately start mechanical positive pressure ventilation with FiO2 0.50, 10 ml/kg tidal volume, and a frequency of 12/min under continuous capnography. Use vet ointment on the eyes to prevent dryness.</li>
		<li>Start balanced anesthesia by continuous intravenous infusion of midazolam 0.5 mg/kg/hr, sufentanil 2.5 &#181;g/kg/hr and pancuronium bromide 0.1 mg/kg/hr. To ensure proper anesthesia continuously monitor ECG, arterial blood pressure, temperature and capnography.</li>
		<li>Intravenously infuse 4.3 mg/kg amiodarone and place the intracardiac defibrillation catheter in the right ventricle using the venous sheeth18.</li>
		<li>Occlude the left anterior descending artery (LAD) distal to the second diagonal branch by intracoronary balloon occlusion, for 90 min, in accordance to previously described protocol18.</li>
	</ol>
	</li>
	<li>Electromechanical mapping
	<ol>
		<li>At four weeks after myocardial infarction, plan the mapping procedure. Prepare the system (Figure 4) in the cathlab for 3D electromechanical mapping (EMM) of the left ventricle. With this system viable, hibernating and infarcted myocardium can be identified without fluoroscopic guidance. To construct such an EM-map acquire&#160;a series of points at multiple locations on the LV endocardial surface by using an ultralow magnetic-field energy source and a sensor-tipped catheter19,20.</li>
		<li>Anesthetize the pig, following protocol steps 4.1.1-4.1.4.</li>
		<li>Place the external reference patch on the pig&#8217;s back.</li>
		<li>Secure vascular access (femoral artery) according to protocol18.</li>
		<li>After obtaining a biplane left ventricular angiogram in the 25&#176; right anterior oblique (RAO) and 40&#176; left anterior oblique (LA) view to estimate left ventricular size, give 75 U/kg of heparin.</li>
		<li>Advance an 8 French-mapping (D or F curve) catheter under fluoroscopic guidance to the descending aorta, aortic arch and across the aortic valve into the left ventricle (LV).</li>
		<li>Orientate the tip of the catheter to the apex of the LV to acquire the first data, followed by outflow tract, lateral and posterior points to form a 3D silhouette, defining the borders of the ventricle.</li>
		<li>Obtain subsequent points until all endocardial segments have been sampled by dragging the mapping catheter over the endocardium and sequentially acquiring the location of the tip while in contact with the endocardium21,22.</li>
		<li>Define the target area, that is where electrical activity is (near) normal and mechanical movement impaired, so-called hibernating myocardium (Figure 6).</li>
	</ol>
	</li>
	<li>Intramyocardial injection
	<ol>
		<li>Replace the mapping catheter by the intramyocardial injection catheter which is composed of a 27-gauge needle and a core lumen inside an 8 French catheter (Figure 5A and B). To deliver specific amounts, load a volume-graded syringe with approximately 2 ml of the hydrogel solution and place it into a syringe pump.</li>
		<li>Adjust the needle extension at 0&#176; and 90&#176; flex and place 0.1 ml of the hydrogel solution to fill the needle dead space. Then, place the injection catheter tip across the aortic valve and into the target area.</li>
		<li>Meet the following criteria for an injection position inside the target area determined in 4.2.9: (1) perpendicular position of the catheter to the LV wall; (2) excellent loop stability (&#60;4 mm) as calculated by the EMM-system; and (3) underlying voltage &#62;6.9 mV21.
		<ol>
			<li>Advance the needle into the myocardium, (4) confirmed by a premature ventricular contraction of the LV, and inject 0.1-0.3 ml of the hydrogel in a bolus at a constant rate of approximately 0.4-0.5 ml/min using the syringe pump. Repeat this at 6-10 different positions as diffuse as possible. The natural pH of the tissue will neutralize the solution after injection, upon which the hydrogel is formed.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Sacrifice
	<ol>
		<li>Post-procedure, humanely sacrifice the animal by exsanguination. Cut the inferior caval vein and remove blood with a suction device. Induce ventricular fibrillation by placing a 9 V battery on the apex.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

A key challenge is to obtain a solution which is injectable through a long catheter while keeping the solution compatible with the bioactive compounds. Although the pH should be increased to increase injectability, bioactive compounds such as growth factors are fragile molecules that should be handled carefully. We monitor the pH of the solution closely using a pH meter after adding the hydrogelator to confirm it is pH 9.0 before adding any bioactive components. Initially, several rounds of adjusting the starting pH of the PBS were necessary to end with the right pH. Furthermore, because we use relatively viscous solutions and a long thin catheter, a large pressure drop is present (in the order of 0.5 MPa, depending on the speed of injection). Therefore, special care should be taken in selecting the right connections between the syringe and the catheter. A syringe pump supports controlled injection, as applying such forces by hand is challenging. For in vitro experiments, the solution was gelated by neutralizing the solution with HCl, while in vivo this is done by the natural pH of the tissue. Therefore, it is important to add the right amount of HCl to prevent an overshoot in pH. The diffusion of this acid is probably the limiting factor in the gelation of the hydrogel in in vitro experiments; however, in vivo the liquid would have a high contact surface area with neutralizing tissue, which will most likely result in a faster and more evenly gelation compared to dropwise addition of concentrated acid. Moreover, the gel switching is much faster with this mild procedure as compared to previously used methods (0.5 hr vs 2 hr)25. Using the body&#8217;s natural pH for switching of the material properties is very appealing since the transition is swift, reversible, cannot occur inside the catheter and is in vivo fully automatic. These properties give advantages over e.g. thermal switchable gels26, where risk of gelation in a catheter due to temperature changes is present, gels that require photo-induced polymerization, which is challenging due to limited light penetration and radical formation27, or gels that require co-injection of a polymerization initiator or accellerator28.
Successful release of a drug from the hydrogel largely depends on the size of the drug. As shown, the small molecule drug is released immediately while the gradual release of the model protein over 1 week shows the promise of these hydrogels as delivery systems for growth factors. In general, hydrogels are more promising as delivery tool for larger objects such as proteins, exosomes and cells29,30.
The 3-D electromechanical mapping and injection procedure provides a clinically validated catheter-based delivery approach for various myocardial regenerative therapies, such as hydrogels. The added value of this technology compared to other non-surgical delivery techniques is the treatment planning, making it possible to differentiate normal, infarcted and hibernating myocardium and to guide therapies in the area of interest. Drawbacks of this approach concern the required technical skills and the time consuming and expensive procedure20. In the presented porcine model of myocardial infarction electromechanical mapping was followed by guided intramyocardial injections with the bioactive supramolecular UPy-hydrogel. Other combinations with regenerative therapies have to be tested in vitro and in vivo to gain more success in this emerging field. Furthermore, optimization of injectability and sterilization procedures have to be performed to successfully translate this method to a clinical setting.

Although the treatment of acute myocardial infarction has significantly improved survival rates, the chronic ischemic heart failure is a major public health problem that progresses with an aging population. There are approximately 6 million heart failure patients in the US with an estimated 25% increase in prevalence in 20301,2. Initial loss of myocardial tissue leads to cardiac remodeling and eventually causes chronic heart failure. Except for heart transplantation, there is no real treatment for this group of patients. The increasing shortcoming of donor hearts emphasizes the need to develop new available therapies to reverse this process of remodeling. Therefore, a goal for future therapies is the regeneration of lost myocardium.
Hydrogels are interesting materials in the field of regenerative medicine because of their biocompatibility, and their sensitivity to external triggers3. Injectable hydrogels offer advantages over non-injectable hydrogels in their use in minimal invasive surgery4. These injectable hydrogels can be applied through a syringe because of their switchability within physiological conditions5, and in principle allow for catheter-based injection approaches6. Different strategies have been used for injectable materials, ranging from chemical crosslinking after injection to physical crosslinking by either temperature, pH and shear-thinning behaviour4,7,8. Although several systems have shown easy injectability via a syringe9,10, full catheter-compatibility has not been shown often6.
Hydrogels prepared from supramolecular polymers are formed by non-covalent interactions which can be switched conveniently from a gel to a solution state, and vice versa using environmental triggers11. Furthermore, the low molecular weight precursors allow for easy processability12,13. The mild conditions required for switching allow the addition of various biological active components such as often difficult to handle growth factors.
Supramolecular transient networks in water based on poly(ethylene glycol) (PEG), end-modified with ureido-pyrimidinone (UPy) moieties14 have shown the benefits of non-covalent interactions in combination with biomedical applications and have been used as drug delivery system in the heart6 and under the renal capsule15. These networks are formed by dimerization of the UPy-groups shielded from the aqueous environment by alkyl spacers forming a hydrophobic pocket. Urea hydrogen bonding facilitates subsequent stacking of these dimers into nanofibers. Due to the reversible interaction of the UPy-UPy dimer, triggers such as pH and temperature can be used to switch from solutions to gels. The use of a synthetic motif allows for design of the molecule and gel properties by for example tuning length of the PEG-chains and alkyl spacers14,16.
Moreover, several bioactive components can be incorporated by simply mixing the supramolecular hydrogelator solution before injection, with drugs or bioactive species, such as growth factors or exosomes, respectively. Exosomes are small membrane vesicles that contain cytosolic derivatives. They are secreted by many cells and are involved in intercellular communication. Exosomes derived from cardiomyocyte progenitor cells are suggested to play a role in cardiac protection17.
Here, we describe the protocol of formulation, and in vivo myocardial injection of such a bioactive supramolecular hydrogel. In vitro experiments are described which give on forehand an indication of gel stability and drug release, which allows for tuning of the gel and release properties before application in vivo.

<table><tbody><tr><td>1 M HCl</td><td></td><td></td><td></td></tr><tr><td>1 M NaOH</td><td></td><td></td><td></td></tr><tr><td>Polystyrene 24-well plate</td><td>Falcon</td><td>353047</td><td></td></tr><tr><td>Amiodarone</td><td>Cordaron I.V. (Sanofini)</td><td></td><td></td></tr><tr><td>Anton Paar Physica MCR501</td><td>Anton Paar GmbH</td><td></td><td>Equipped with a parallel-plate geometry (25 mm)</td></tr><tr><td>Atropine</td><td>PCH</td><td></td><td></td></tr><tr><td>Balloon ventilator</td><td></td><td></td><td></td></tr><tr><td>Cary 50 Scan UV-Visible Spectrophotometer</td><td>Varian</td><td></td><td></td></tr><tr><td>Cary Eclipse Fluorescence Spectrophotometer</td><td>Varian</td><td></td><td></td></tr><tr><td>Defibrillation patches</td><td></td><td></td><td></td></tr><tr><td>DMSO</td><td>Biosolve</td><td>44705</td><td></td></tr><tr><td>Endotracheal tube</td><td>Covidien</td><td></td><td></td></tr><tr><td>Heparin</td><td></td><td></td><td></td></tr><tr><td>Ketamine</td><td>Narketan 10 V&amp;eacute;toquinol</td><td></td><td></td></tr><tr><td>Mapping catheter 115&amp;nbsp;cm</td><td>Biosense Webster</td><td></td><td></td></tr><tr><td>Midazolam</td><td>Actavis</td><td></td><td></td></tr><tr><td>MilliQ</td><td>MD Milipore MilliQ Integral Water Purification System</td><td></td><td></td></tr><tr><td>mRuby2</td><td></td><td></td><td></td></tr><tr><td>NaCl 0.9% 500&amp;nbsp;cc</td><td>Braun</td><td></td><td></td></tr><tr><td>NOGA guided Myostar injection catheter</td><td>Biosense Webster</td><td></td><td></td></tr><tr><td>NOGA-RefStar EFO-patch</td><td>Biosense Webster</td><td></td><td></td></tr><tr><td>Pancuronium bromide</td><td></td><td></td><td></td></tr><tr><td>Parafilm</td><td>VWR</td><td>IKAA3801100</td><td></td></tr><tr><td>PBS</td><td>Sigma Aldrich</td><td>P4417&amp;nbsp;</td><td></td></tr><tr><td>PET millicel</td><td>Millipore</td><td>PIEP12R48</td><td></td></tr><tr><td>Pirfenidone</td><td>Sigma Aldrich</td><td>P2116</td><td>Used from 100&amp;nbsp;mM stock in DMSO</td></tr><tr><td>Sodiumthiopental</td><td>Inresa</td><td></td><td></td></tr><tr><td>Sufentanil</td><td>Sufentanil-Hameln</td><td></td><td></td></tr><tr><td>Tegaderm</td><td></td><td></td><td></td></tr><tr><td>UPy-PEG10k</td><td></td><td></td><td></td></tr><tr><td>UV-Lamp</td><td></td><td></td><td></td></tr><tr><td>Vet ointment</td><td></td><td></td><td></td></tr><tr><td>Visipaque contrastfluid 100&amp;nbsp;cc</td><td></td><td></td><td></td></tr></tbody></table>

an injectable and drug-loaded supramolecular hydrogel for local catheter injection into the pig heart

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Typical results obtained from the oscillatory rheological measurements on both the solution and the gel are shown in Figure 1. For injection through a long catheter, a Newtonian fluid with low viscosity is desirable. Viscosity was measured as function of shear rate, showing that at pH 8.5 the solution is shear thinning but at pH 9.0 and 9.5 the solutions behave as Newtonian fluids as evidenced by the constant viscosity of 0.54 and 0.36 Pa&#183;sec, respectively (Figure 1A). After neutralizing the samples, the samples show a solid-like response observed by a storage modulus G&#8242; which is larger than the loss modulus G&#8243; and therefore a tan&#948; = G&#8243;/G&#8242; &#60;1 (Figure 1B). The gel obtains its final strength within 30 minutes. Oscillatory rheological measurements show a typical solid-like response with G&#8242; almost independent of the angular frequency and G&#8242; &#62;G&#8243; for all frequencies measured (Figure 1C).
Essential for the use as drug delivery system is the erosion of the hydrogel over time. The supramolecular interactions are inherently dynamic and allow for a slow erosion of the gel in vitro. Erosion and release experiments are performed at 37 &#176;C using porous well inserts (Figure 2A and B). By tuning the length of the hydrophobic and hydrophilic block14, a gel that erodes over a period of several weeks can be obtained (Figure 3A). The gel erodes 25% in 2 weeks with an initial erosion of 10% in the first day, presumably due to initial swelling of the hydrogel. As example, both the release of a small molecule drug (pirfenidone), and the release of a model fluorescent protein (mRuby2) was studied. A fluorescent model protein allows for an easy readout; however, in vitro release experiments can also be performed on other proteins using ELISA for quantification6. The small molecule drug is released within a day, while bigger molecules such as proteins are gradually released over 1 week (Figure 3B). Fitting the release profile of mRuby2 up to 60% release with the semi-empirical Korsmeyer-Peppas model indicates release due to diffusion (n = 0.44)23. The absence of an offset in the (adapted) Korsmeyer-Peppas model shows that there is no burst release present for mRuby224. Because of the limited amount of data points with a release lower than 60% for pirfenidone, no fitting was performed on this release profile.
The catheter navigation system consists of a communication unit console, a workstation (Figure 4), a triangular location pad (generating a low magnetic field) with an external reference patch, and two catheters, the sensor-tipped mapping and the injection catheter (Figure 5).
After post-processing analysis has filtered unstable points the 3D endocardial reconstruction of the LV is updated in real time with the acquisition of each new data point and is continuously displayed as unipolar and bipolar voltage potentials on a graded color scale (Figure 6A). The local linear shortening (LLS) function quantifies regional wall motion by obtaining the average change in distance between sample site and adjacent points at end-systole and end-diastole. The mean voltage and LLS values are calculated for each segment and displayed in the polar map. (Figure 6B). The presence of an abnormal or low unipolar potential (&#8804;6 mV) and impaired mechanical activity (LLS &#8804;4%) characterizes infarcted areas22.
<img alt="Figure 1" src="/files/ftp_upload/52450/52450fig1highres.jpg" width="700" /> 
Figure 1:. Rheological assessment of the solutions and gels. (A) Viscosity as a function of shear rate for the solutions at different pH. For the sample at pH 8.5 shear thinning is observed but for the samples at pH 9.0 and 9.5 constant viscosities are obtained, showing the Newtonian behavior of these solutions. (B) Gel curing followed by plotting tan &#948; as a function of time. (C) Frequency sweep for a neutralized sample after 2 hr curing. Error bars show standard deviations of 3 independent measurements, indicating a typical experimental error.
<img alt="Figure 2" src="/files/ftp_upload/52450/52450fig2highres.jpg" width="600" /> 
Figure 2: Setup for degradation and release experiments. (A) Poly(ethylene terephthalate) well insert covered with Parafilm to prevent leakage during preparation. (B) 24-wells plate with inserts, wrapped with Parafilm to prevent evaporation of the solvent.
<img alt="Figure 3" src="/files/ftp_upload/52450/52450fig3highres.jpg" width="700" /> 
Figure 3: Erosion and Release. (A) Erosion of the hydrogel over time. Gradual erosion of the gel for at least 2 weeks is observed. (B) Release of a small molecule drug and a model protein. While the small molecule is released within a day, the model protein is gradually released over a week without a significant burst release. The line shows the fit of the Korsmeyer-Peppas model to the initial stage of the release.
<img alt="Figure 4" src="/files/ftp_upload/52450/52450fig4highres.jpg" width="600" /> 
Figure 4: The catheter navigation system. Communication unit console with NOGA XP Cardiac Navigation System.
<img alt="Figure 5" src="/files/ftp_upload/52450/52450fig5highres.jpg" width="700" /> 
Figure 5: (A) The intramyocardial injection catheter with syringe attached. (B) Detail of injection needle.
<img alt="Figure 6" src="/files/ftp_upload/52450/52450fig6highres.jpg" width="700" /> 
Figure 6: Unipolar voltage and LLS map. (A) Unipolar map, LAO view (top) and bulls eye (below). Red color indicates low unipolar voltage values at myocardial base (normal) with loss of electrical activity posterolateral. Blue indicates normal myocardium, whilst green and yellow colors indicate decreased viability. (B) LLS map, LAO view (top) and bulls eye (below). Red color indicates akinesia in the posterolateral wall, green and yellow indicate decreased wall motion. The mapping points are shown by white dots. The drawn white line shows the area of interest, characterized by decreased unipolar voltages and impaired wall motions. Brown points represent the injection sites.

Watch this Scientific Journal Video about An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart at JoVE.com

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

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Research

JoVE Journal

Bioengineering

17.3K Views.  Eindhoven University of Technology.  Supramoleculaire hydrogelators gebaseerd op ureido-pyrimidinonen maken volledige controle over de gel macroscopische eigenschappen en de sol-gel-schakelgedrag met pH. Hier presenteren we een protocol voor de formulering en het injecteren van een dergelijke supramoleculaire hydrogelator via een katheter afgiftesysteem voor lokale afgifte direct betrokken gebieden in het varkenshart. 

Video: An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Medicine

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are composed of two components: hydrophobically modified cellulose as the network polymer and self-assembled core-shell nanoparticles that act as non-covalent cross linkers through dynamic, multivalent interactions. These methods describe both the formation of these self-assembled nanoparticles through nanoprecipitation as well as the formulation and mixing of the two components to form hydrogels with tunable mechanical properties. The use of dynamic light scattering (DLS) and rheology to characterize the quality of the synthesized materials is also detailed. Finally, the utility of these hydrogels for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery is demonstrated through in vitro experiments to characterize&#160;drug release, thermal stability, and cell settling and viability. Due to its biocompatibility, injectability, and mild gel formation conditions, this hydrogel system is a readily tunable platform suitable for a range of biomedical applications.

This protocol describes the synthesis and formulation of injectable, supramolecular polymer-nanoparticle (PNP) hydrogel biomaterials. Applications of these materials for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery are demonstrated.

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Novel Percutaneous Approach for Deployment of 3D Printed Coronary Stenosis Implants in Swine Models of Ischemic Heart Disease

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally (3D) printed coronary implants can be employed to percutaneously create a focal coronary stenosis. However, reliable delivery of the implants can be difficult without the use of ancillary equipment. We describe the use of a mother-and-child coronary guide catheter for stabilization of the implant and for effective delivery of the 3D printed implant to any desired location along the length of the coronary vessel. The focal coronary narrowing was confirmed under coronary cineangiography and the functional significance of the coronary stenosis was assessed using gadolinium-enhanced first-pass cardiac perfusion MRI. We showed that reliable delivery of 3D printed coronary implants in swine models (n = 11) of ischemic heart disease can be achieved through repurposing mother-and-child coronary guide catheters. Our technique simplifies the percutaneous delivery of coronary implants to create closed-chest swine models of focal coronary artery stenosis and can be performed expeditiously, with a low procedural failure rate.

We describe a novel, cost-effective, and efficient technique for percutaneous delivery of three-dimensionally printed coronary implants to create closed-chest swine models of ischemic heart disease. The implants were fixed in place using a mother-and-child extension catheter with high success rate.

Minimally invasive methods for creating models of focal coronary narrowing in large animals are challenging. Rapid prototyping using three-dimensionally ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...

Delivery of Cardioactive Therapeutics in a Porcine Myocardial Infarction Model

Myocardial infarction is one of the leading causes of death and disability worldwide, and there is an urgent need for novel cardioprotective or regenerative strategies. An essential component of drug development is determining how a novel therapeutic is to be administered. Physiologically relevant large animal models are of critical importance in assessing the feasibility and efficacy of various therapeutic delivery strategies. Due to their similarities to humans in cardiovascular physiology, coronary vascular anatomy, and heart weight to body weight ratio, swine is one of the preferred species in the preclinical evaluation of new therapies for myocardial infarction. The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. After percutaneously induced myocardial infarction, female landrace swine received treatment with novel agents through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump. The procedures employed for each technique are reproducible, resulting in reliable cardioactive drug delivery. These models can be easily adapted to suit individual study designs, and each of these delivery techniques can be used to investigate a variety of possible interventions. Therefore, these methods are a useful tool for translational scientists pursuing novel biological approaches in cardiac repair following myocardial infarction.

The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. Female landrace swine received treatment through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump.

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

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