Cardioprotection of PLGA/gelatine cardiac patches functionalised with adenosine in a large animal model of ischaemia and reperfusion injury: A feasibility study

Abstract The protection from ischaemia‐reperfusion‐associated myocardial infarction worsening remains a big challenge. We produced a bioartificial 3D cardiac patch with cardioinductive properties on stem cells. Its multilayer structure was functionalised with clinically relevant doses of adenosine. We report here the first study on the potential of these cardiac patches in the controlled delivery of adenosine into the in vivo ischaemic‐reperfused pig heart. A Fourier transform infrared chemical imaging approach allowed us to perform a characterisation, complementary to the histological and biochemical analyses on myocardial samples after in vivo patch implantation, increasing the number of investigations and results on the restricted number of pigs (n = 4) employed in this feasibility step. In vitro tests suggested that adenosine was completely released by a functionalised patch, a data that was confirmed in vivo after 24 hr from patch implantation. Moreover, the adenosine‐loaded patch enabled a targeted delivery of the drug to the ischaemic‐reperfused area of the heart, as highlighted by the activation of the pro‐survival signalling reperfusion injury salvage kinases pathway. At 3 months, though limited to one animal, the used methods provided a picture of a tissue in dynamic conditions, associated to the biosynthesis of new collagen and to a non‐fibrotic outcome of the healing process underway. The synergistic effect between the functionalised 3D cardiac patch and adenosine cardioprotection might represent a promising innovation in the treatment of reperfusion injury. As this is a feasibility study, the clinical implications of our findings will require further in vivo investigation on larger numbers of ischaemic‐reperfused pig hearts.

Despite decades of impressive improvements in therapies for patients developing MI, the current therapies do not fully restore the functionality of the damaged myocardial tissue. Paradoxically, about 50% of the myocardium damage after MI is a result of the rapid reoxygenation of the tissues, known as reperfusion injury (Hausenloy & Yellon, 2008). Reperfusion injury therefore remains a major problem in the treatment of MI patients.
However, the postconditioning translation into clinical practice has so far been disappointing. Moreover, the pharmacological agents suggested for infarct size limitation have serious side effects when used at cardioprotective doses, which further hinders their clinical use (Pagliaro & Penna, 2014;Sanada, Komuro, & Kitakaze, 2011). One solution to the problem might be a timely and direct delivery of the cardioprotective drugs into ischaemic-reperfused myocardium, in order to administer the drugs in a sufficient concentration to achieve the highest efficiency and limit the side effects (Ye & Yang, 2009).
There is now considerable evidence for adenosine involvement in the mechanisms of cardiac protection from conditioning-induced ischaemia. In particular, by using adenosine and selective agonists and antagonists of several adenosine receptors (Donato et al., 2007;Norton, Jackson, Turner, Virmani, & Forman, 1992;Xi et al., 2009), as well as genetic approaches (Morrison, Tan, Ledent, Mustafa, & Hofmann, 2007;Xi et al., 2008), it has been clearly demonstrated that adenosine and adenosine receptors activation plays a role of paramount importance in cardioprotection against ischaemia/reperfusion myocardial injury. The current thought is that all the adenosine receptors are needed and interact to produce cardioprotection (Lasley, Kristo, Keith, & Mentzer, 2007;Urmaliya, Pouton However, the clinical use of adenosine for myocardial protection has yielded conflicting results and may be limited because of its heavy side effects, such as hypotension and bradycardia (Marzilli, Orsini, Maraccini, & Testa, 2000). It is therefore likely that the modality of drug delivery may be crucial.
We recently produced bioartificial cardiac patches and demonstrated their micropatterning-mediated effect to guide and control proliferation and differentiation of stem cells in cardiac sense (Cristallini et al., 2014;Cristallini et al., 2016).
In this research, we investigated on a small number of pigs (n = 4) the potentiality of multilayered Poly(lactic-co-glycolic acid) (PLGA)/gelatine cardiac patches for the targeted delivery of adenosine into the ischaemic-reperfused large-mammalian heart. The primary goal of this feasibility study was to compare the differences between functionalised and nonfunctionalised patch. Although the exact cardioprotective mechanisms of adenosine are not yet fully understood, we studied the effects of adenosine on some protective/prosurvival kinases (Ovize et al., 2010). The physicochemical, degradative, and pharmacological characterisation of the scaffolds was performed before and after implantation. Furthermore, histomorphologic, biochemical, and spectroscopic analyses of the explants were carried out, to verify the cardioprotective effect of the adenosine-loaded patch.
The functionalisation method with adenosine (>99%, Sigma Aldrich, USA) was optimised ( Figure 1a) in order to reach an immediate and quantitatively elevated release of adenosine in a very short time, as described in Supporting Information. Identical patches, but without adenosine functionalisation, were also produced (MMP). The patches were dried and sterilised according to a previously optimised method (Rosellini, Cristallini, Barbani, Vozzi, & Giusti, 2009 (1-3_AMI) and one after 3 months of follow-up (4_AMI) ( Table 1).
1_AMI was used to set up the surgical technique, to test the proper number of biopsy samples, and to verify the presence of ischaemic areas after left anterior descending (LAD) coronary artery ligature.
To assess the feasibility of patch application on a beating heart and the effect of adenosine on the acute myocardial infarct model, two pigs were sacrificed at 24 hr after implantation of the patches, one  Note. 1_AMI: animal subjected to LAD to induce ischaemia, follow up at 24-hr acute myocardial infarct model; MMP: multilayer microstructured patch (patch nonloaded with adenosine); ADMMP: adenosine delivery multilayer microstructured patch (patch loaded with adenosine); 2_AMI + MMP: animal subjected to LAD to induce ischaemia, follow up at 24 hr acute myocardial infarct model treated with patch nonloaded with adenosine (MMP); 3_AMI + ADMMP: animal subjected to LAD to induce ischaemia, follow up at 24 hr acute myocardial infarct model treated with patch loaded with adenosine (ADMMP); 4_CMI + ADMMP: animal subjected to LAD to induce ischaemia, follow up at 3 months chronic myocardial infarct model treated with patch loaded with adenosine (ADMMP). unloaded (2_AMI + MMP) and one loaded with adenosine (3_AMI + ADMMP). To evaluate patch bioabsorbance and preliminary patch effect at a medium follow-up, one pig was sacrificed at 3 months after the implantation of an adenosine-loaded patch (4_AMI + ADMMP). Details concerning surgical procedures and bioptic sampling can be found in Supporting Information. In short, a biopsy was performed 10 min before the LAD occlusion in the area in which ischaemia would have later been induced. For all biopsies, 14G disposable guillotine needles were used. The LAD occlusion was performed by interrupting the anterior descending coronary blood flow with a tourniquet. Five minutes after the LAD ligature, the patch was sutured on the ischaemic area of the myocardium and fixed with four stitches of 5.0 prolene. Two biopsies, one in the ischaemic area and one in a remote area, were performed 45 min after LAD occlusion. After 50 min from the occlusion, the tourniquet was released (LAD reopening, reperfusion step), and 10 min later, two myocardial biopsies, one in the ischaemic area and one in a remote area, were performed. Finally, two additional biopsies, one in the ischaemic area and one in a remote area, were performed 60 min after LAD reopening; the surgical cut was then sutured, and the animal was awakened. The animals were followed daily to measure sensitive parameters such as sensorium, conjunctival mucous membranes, body temperature, ventilation rhythm and breathing rate, and heart rate. Daily dressing of surgical wounds was performed. In the early days of follow-up, the animal was kept under drug therapy that included the use of morphine, analgesics (FANS), and antibiotics.

| Histomorphological analysis
After the animal sacrifice, the heart was explanted and analysed from a macroscopic and microscopic point of view. The heart was observed, and pictures were taken using a stereoscopic microscope (Leica Z6 APO) to highlight the presence of macroscopic interactions between the patch and the myocardium. At the end of the macroscopic examination, a sample of the myocardium underlying the patch was taken for histological analysis, and a sample of the myocardium in an area not interested by the ischaemic event was taken as a control.
The obtained samples were processed for paraffin embedding.
The samples were sliced in 4-to 5-μm serial sections and stained with haematoxylin/eosin (HE) and Gomori stains. The slides were observed and photographed under a biological microscope (Nikon Eclipse E600). Ten pictures per animal, at 40× magnification, were obtained from the HE-stained serial sections. The pictures were taken randomly in the area immediately under the patch. The epicardial thickness was measured using the image analysis software Image-Pro Premier 9.1 (Media Cybernetics). Ten linear measures were performed on each sample (2_AMI + MMP, 3_AMI + ADMMP, and 4_AMI + ADMMP) and the epicardial thickness calculated as the mean of the obtained values. Inflammatory cells (macrophages, eosinophils, neutrophils, lymphocytes, and mastocytes) were distinguished based on their morphology and dye affinities. The total cell number and the inflammatory cell number were accurately counted using the Image-Pro Premier 9.1 software. Other samples, both underlying the patch and in a remote area, were taken and frozen for physicochemical analyses.

| Biochemical study of the biopsies by means of western blot analysis
Western blot was used to analyse the heart biopsies. Specific antibodies against porcine total AKT (diluted 1:1000), phosphorylated Akt (diluted 1:1000), total ERK 1/2 (diluted 1:1000), phosphorylated ERK 1/2 (Diluted 1:500; all from Cell Signaling Technology, USA), and α-actinin (1:10000, Sigma-Aldrich, USA) were used. Expression levels of phospho-AKT and phospho-ERK 1/2 were normalised to both α-actinin and total AKT/total ERK proteins. For each animal, the expression levels of phospho-AKT and phospho-ERK proteins detected in the biopsies, sampled after either LAD ligature or LAD reopening, were normalised against the expression levels detected in the biopsies taken before LAD ligature, to reduce intertest animal biological differences.
2.5 | In vitro morphological, physico-chemical, and functional ADMMP characterisation before and after apposition onto myocardium The attenuated total reflectance (ATR) sampling technique was performed on small fragments of ADMMP or on myocardial samples.
Spectral images were acquired in μATR mode (the spectral resolution was 4 cm −1 , and the spatial resolution was 100 × 100 μm). The spectra were collected by putting in contact the ATR objective and the sample and collecting the spectrum generated from the surface layer of the sample. The obtained correlation map indicates the areas of each image where the spectra are most similar to a reference spectrum. The band ratio analysis involves the measurement of the band intensity or area of an internal reference, with respect to that of a band of interest.
FT-IR analysis was carried out directly on the patch detached from 2_AMI + ADMMP and 3_AMI + ADMMP tissues after 24 hr of followup. The analysis was not feasible on the patch from the 4_AMI + ADMMP tissue, because the patch was completely reabsorbed after 3 months. A FT-IR analysis was carried out on myocardium samples both from the ischaemic area and remote areas after 24 hr and 90 days of follow-up. The myocardium samples underlying the patch were further sliced, obtaining closer (proximal) and deeper (distal) sections respect to patch positioning. Before FT-IR analysis, all samples were lyophilised at the same temperature rate.
FT-IR chemical imaging allows to gain information about the chemical composition of healthy and diseased human tissues (Liu et al., 1996;Liu, Dixon, & Mantsch, 1999); the most important parameters to distinguish acute and chronic MI are reported in Table 2.

| In vitro characterisation of ADMMP before implantation
After sterilisation, ADMMP was subjected to the in vitro release test showing a high delivery in the interval from 1 min to 1 hr (Figure 1 b), corresponding to 43% with respect to the amount contained in the device (17.6 ± 0.2 mg), in line with the doses of intraluminally administered adenosine (Ross et al., 2005).
SEM images of ADMMP before and after adenosine release (Figure 1c-f) and spectra obtained by FT-IR chemical imaging confirmed that the adenosine was completely released as described in Supporting Information.

| Histomorphological analyses
The hearts were explanted at either 24 hr or 3 months follow-up. The Histomorphometrical analysis showed that the total cell number was considerably higher in 2_AMI + MMP than 3_AMI + ADMMP, and the number of inflammatory cells was, respectively, 62% and 38% of the total number. Furthermore, in 2_AMI + MMP, mononucleates seemed to outnumber polymorphonucleates, whereas in 3_AMI + ADMMP, most of the inflammatory cells were polymorphonucleates, mostly eosinophils. All these findings suggested protective effects of adenosine-loaded patches.

| Biochemical study
To dissect the putative mechanisms of the cardioprotective effects of adenosine-loaded patches, we quantified the RISK pathway activation on the heart biopsies taken at 0-10-60 min time course after LAD.
The left ventricular (LV) tissue from the non-infarcted zone (remote area biopsies) showed no change in phospho-ERK and phospho-AKT expression (data not shown). However, if compared with the 2_AMI + MMP animal that did not show any significant signs of activation, LV tissue sampled close to the ischaemic area from 3_AMI + ADMMP and 4_AMI + ADMMP showed RISK pathway activation. An increase in AKT phosphorylation was highlighted in 3_AMI + ADMMP, even in the biopsy taken before LAD reopening  3.5 | Physico-chemical analysis on myocardial tissue by means of FT-IR chemical imaging The GAG/protein ratio at the interface with the patch was evaluated in the myocardial samples of 2_AMI + MMP and 3_AMI + ADMMP and compared with the GAG/protein ratio in a remote area of the same animal (control; Figure 4a,b). No substantial differences were found between the GAG/protein ratio for myocardial 3_AMI + ADMMP samples taken at the level of the proximal (R = 0.23-0.30) and distal section (R = 0.24-0.33). These results are in line with the value obtained from a control area (R = 0.29). As to 2_AMI + MMP myocardial tissue, a significant variability of the GAG/protein ratio (R = 0.18-0.44), indicative of GAG component degradation (low values) and interstitial oedema (high values), can be observed. In Figure 4c-e, the chemical maps of tissues sampled at proximal and distal level are reported and compared with that of the control tissue. The mean values measured for the proximal area (R = 0.23-0.28) and for the distal area (R = 0.23-0.28) are only in limited domains slightly lower than the one measured for healthy tissue (R = 0.29). This reduction is likely not due to a fibrosis condition (a process that can last 12 months after the injury) but to the presence of collagen deposition areas, an event that is part of the normal wound healing process.
For 4_AMI + ADMMP tissue, CH 2 /CH 3 stretching peak ratio, GAG/protein ratio, and amide deconvolution were the considered parameters. The cellularisation of the tissue was evaluated by CH 2 / FIGURE 5 FT-IR chemical imaging spectra, second derivative spectra, and corresponding chemical map: (a) CH 2 /CH 3 stretching peak ratio for control healthy tissue, (b) for myocardial samples of 4_AMI + ADMMP in the correspondence of proximal area, and (c) distal area respect to ADMMP position [Colour figure can be viewed at wileyonlinelibrary.com] CH 3 stretching peak ratio also for 4_AMI + ADMMP. In Figure 5, second derivative spectra and corresponding chemical maps for a healthy tissue and for the myocardial tissue of 4_AMI + ADMMP acquired at both proximal and distal levels are reported. It is important to state that the CH 3 stretching band can be attributed to the presence of membrane phospholipids, whereas the CH 2 stretching peak derived from glycine and proline residues of collagen. The CH 2 /CH 3 (2,926 cm −1 /2,956 cm −1 ) peak ratio can therefore be considered as indicative of both cellularisation and collagen deposition.
As evidenced by the comparison of ratio values corresponding to R = 2.45-2.80 for an healthy tissue, R = 2.10-2.45 for tissue at proximal level, and R = 2.10-2.80 for the tissue at distal level, no substantial variations in the ratio are observed with respect to the control tissue, indicating that there is no significant decrease of cells and/or increase of collagen in the tissue that was in contact with the device after 3 months.
In Figure 6, the second derivative spectra are reported, acquired from the maps of tissue after 3 months of contact with the ADMMP at proximal level and compared with those acquired from a negative control (healthy myocardial tissue) and from a positive control (infarcted tissue). In the spectrum of negative control (Figure 6a), typical peaks of a healthy tissue can be observed. In Figure 6b, the second derivative spectrum from a region involved in an infarction process was reported. Substantial differences are evident respect to the spectrum from healthy tissue; in particular, it can be observed a reduction of intensity of the bands at 1,740-1,720 cm −1 , reflecting phagocytosis of membrane lipids and nucleic acid after infarction. In addition, an intense absorption of the amide I at 1,643 cm −1 , typical of the infarcted tissue, was revealed. Furthermore, the adsorption at 1,655 cm −1 and at 1,629 cm −1 due to α-helical and β-turn protein structure, respectively, is reduced in infarcted heart tissue.
In Figure 6c, the spectrum of the proximal area at the interface with the functionalized patch was reported. The main absorption of amide I at 1,643 cm −1 that appears in the infarcted tissue is not significant in the area in contact with the device. In addition, the band at 1,650 cm −1 is more intense than that at 1,655 cm −1 , suggesting a deposition of newly synthesised collagen. Particularly, significant is the absence, in the proximal area, of the band absorption at 1,638 cm −1 , relative to the triple helical structure of collagen in fibrotic conditions. The presence of bands in the 1,740 to 1,720-cm −1 range is highly significant for a good re-cellularisation of post-infarction tissue.

| DISCUSSION
The present work represents a feasibility study for the employment of an innovative PLGA/gelatine cardiac patch functionalised wth adenosine for cardioprotective purposes in a large-animal model.
On the basis of our previous encouraging results with microstructured (Cristallini et al., 2014) and multilayer (Cristallini et al., 2016) patches for stem cell differentiation, the present study was designed to expand our knowledge on a functionalized PLGA/gelatine cardiac patch as drug carrier in a large-animal model in order to validate the novel paradigm of controlled adenosine delivery to reduce myocardial reperfusion injury, using a patch able also to guide the cardiac tissue regeneration.
Physico-chemical and morphological analyses confirmed the validity of the used loading method, showing how the assembling of the scaffold represents an optimal platform for a quick and sustained release of the cardio-protective agent. After completion of the drug reservoir function, reducing the reperfusion injury, the patch can continue to carry out its second function, which consists in driving the regenerative processes, as already described in our previous work (Cristallini et al., 2014;Cristallini et al., 2016).
In vitro and in vivo tests indicate that the amount of adenosine in the intermediate layer is completely released. In particular, in vivo, at 24 hr of follow-up, adenosine was completely released from the patch, whereas part of the drug was still present in the tissue underneath the patch and not yet metabolised. Remarkably, the dose of adenosine that we used, about 11 mg within the myocardial area at risk (corresponding to about 20 g of myocardium), was concentrated and present in the tissue for a long time. This dose therefore represents several times the usual dose (10-12 mg/kg for the entire body) intravenously infused for 3 hours (Ross et al., 2005).
A series of advantages for our delivery system can therefore be considered as compared with direct injection: (a) It allows an early application of adenosine, which will be present at the moment of reperfusion when the cardioprotective mechanisms must be already However, the transition to the clinical arena has been fairly successful.
Nevertheless, and despite the controversial results with adenosine, this endogenous factor resulted superior to other drugs in inducing cardioprotection in several clinical studies (Niccoli et al., 2013;Singh et al., 2012). Here, the promising protective effect of very high doses of adenosine, with no evidences of side effects, is corroborated by the improvement of other signs and features of the lesion and by the wellbeing of the treated animals.
It has been recently demonstrated that the microscopic morphology and the chemical composition of the tissue are particularly relevant for MI progression (Colley, Kazarian, Weinberg, & Lever, 2004;Yang et al., 2011). In this work, the use of FT-IR allowed to gather specific chemical information on different components within an MI site, discriminating between altered and healthy regions and pointing out the effects of a patch both loaded with adenosine (ADMMP) and not loaded (MMP) on the infarcted areas. One important advantage is the possibility to carry out the analysis without the need of staining the area of interest, allowing obtaining information about molecular composition on heterogeneous substrates, such as biological samples (Yang et al., 2011). Altogether, this and other studies (Gough, Zelinski, Wiens, Rak, & Dixon, 2003;Liu et al., 1996;Liu et al., 1999;Samouillan et al., 2017) confirm that FT-IR chemical analysis can be used to evaluate pathological changes in MI in a very short time, and the results are consistent with the infarction fibrosis region shown with HE staining. The possibility to complement histological and biochemical analyses with the FT-IR chemical imaging technique increases the number of investigations while limiting the number of animals to be employed; this is very important as it impinges upon the highly desirable accomplishment of the 3Rs (reduce, refine, and replace) rule in bio-nanomedicine applications (Accomasso, Cristallini, & Giachino, 2018).
The histological, histomorphometrical, and FT-IR analyses carried out at the level of the tissue/patch interface provided a picture of a tissue in dynamic conditions, in which normal cardiomyocytes are associated to the biosynthesis of new collagen and to a less-fibrotic outcome of the healing process in hearts treated with functionalised patches.
Due to the low number of analysed samples, this represents a feasibility study for an innovative and unconventional approach to cardiovascular disease, and the clinical implications of these findings will require further investigation. Concerning the pharmacological aspect, several groups of drugs with different mechanisms of action might potentially be targeted to the heart using PLGA/gelatine cardiac patches (e.g., bradykinin, opioids, insulin-like growth factor-1, transforming growth factor-β1, cyclosporine A, and nicorandil).
Indeed, a widespread clinical use of these drugs at the required doses for a cardioprotective response was hampered by the high risk of dangerous side effects. The targeted delivery strategy proposed here may help to overcome these limitations.
In conclusion, a PLGA/gelatine 3D cardiac patch was functionalised with adenosine in order to obtain a synergistic effect between patch cardio-inductivity and adenosine cardioprotection, a previously unexplored combination at the basis of a recent international patent (PCT/IB2014/0580025). The major finding is that the functionalisation of bilayered PLGA/gelatine cardiac patches with adenosine enables its targeted delivery to the ischaemic-reperfused area of the heart, where it promotes several beneficial effects. Besides the activation of pro-survival signalling pathways, the patch functionalised with adenosine apparently promotes a more pronounced cellularisation at 24 hr follow-up and a non-fibrotic outcome at 3 months follow-up.
Vaccari for the design and acquisition of data; N. Barbani, E. Cibrario Rocchietti, R. Barberis, M. Falzone, K. Cabiale, G. Perona, E. Bellotti and R. Rastaldo for the acquisition, analysis, and interpretation of data; S. Pascale for the design and interpretation of data; and P. Pagliaro for the interpretation of data and paper drafting.

SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of the article.