Hydrogel-based delivery for antineoplastic drugs and vascular scaffolding

Hydrogels, hydrophilic polymeric compounds, have been recently put under investigation as regenerative medicine applications and delivery systems for antineoplastic drugs, particularly chemotherapeutics (anthracyclines, alkylating agents), target drugs (trastuzumab) and immunotherapies. Porosity, conductivity, biodegradability and physical states are some of the peculiarities that render hydrogels suitable for therapies implementation. Chemically-modifying agents and enzymes can be also coupled to hydrogels for pharmacokinetical parameters improvement and side effects avoidance. Cardiotoxicity is in fact one of the major issues for oncological patients after treatment efficacy. Heart failure, myocarditis and hypertension are causes of morbidity and mortality that can possibly be avoided. Specific reaching of the target tumor site has been achieved by several authors in preclinical in vivo studies but clinical Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 6 September 2021 doi:10.20944/preprints202109.0104.v1 © 2021 by the author(s). Distributed under a Creative Commons CC BY license. studies are currently under design processes. Polydioxanone, a hydrogel-mimicking agent, is capable to interact with the elastic properties of pulmonary artery. An advantageous characteristic is that can be also reabsorbed within biological systems and can cause a remodeling process of the vessel wall. Hydrogels currently represent a strong topic of interest for researchers and probably will guide future clinical investigations and practice.

for creating biocompatible scaffolds that can mimic the action of the endogenous matrix, promote cell growth and repair target tissue [14].

Structural attributes and manifacturing dissimilarities
Hydrogels are three-dimensional, highly hydrophylic polymeric compounds: they are able to absorb a large amount of fluids, mainly water (90-99%), exchanging oxygen and increasing in volume without altering their composition [14,15]. Morevoer, the high fluid content makes them suitable to absorb and encapsulate cells [10]. Hyrogels were first used by Wichterle and Lim, who developed a cross-linked hydrophilic polymer (2-hydroxyethyl methacrylate) to produce contact lenses [15].
Depending on the composition of the polymers, hydrogels can be classified into: -Homopolymers, if derived from a single monomer species, -Copolymers, two or more monomer species with at least one hydrophilic component, -Multipolymer Interpenetrating polymeric hydrogel (IPN), consisting of two polymers, natural and/or synthetic, crosslinked [16].
Other elements that characterize hydrogels are porosity (size of the pores, distribution), biodegradability, conductivity, density and cross-linking (figure 1) [10,17]. Combined together, they define structural and biological properties of these compounds.
Porosity, permeability and mechanical properties of scaffolds are important factors that condition cell growth, migration and colonization. The porosity of a scaffold is given by the presence of pores, open and closed, of different sizes, shapes, spatial distribution and interconnection [15]. Therefore, by changing the composition, the density of the cross-linking, the polymerization conditions, and the mechanical properties of hydrogel can be modified [15].
The hydrogel manufacturing process is also important because it will determine its characteristics, such as the size of the pores, their distribution and the forces of coexistence.
On the basis of the composition of hydrogel materials they can be distinguished in naturalbased, synthetic-based and semisynthetic-based [10,16]. In general, synthetic materials are hydrophobic in nature and have a greater mechanical strength that reduces degradability and increases durability of the material [16]. Natural-based hydrogels are usually more biocompatible and biodegradable [18]. They possess peptide sequences that are recognized by membrane receptors, increasing interaction with cells and consequently cell proliferation [18].
The major limitation linked to the use of natural materials is their lack of availability, as well as the poor ability to control degradation [19,20].
Natural compounds commonly used are fibrin, collagen, hyaluronic acid.
Synthetic-based hydrgels are easy to produce, which results in greater standardization and control of physical characteristics such as conductivity, contractivity, density, porosity [10,21].
However, synthetic materials lack recognizable portions that can develop cellular responses [10]. Synthetic materials can be furhter classified into non-degradable and biodegradable, depending on their ability to maintain intact their physical characteristics or be degraded [10].
Among them, poly ethylene-glycols (PEG), stands out for its versatility and extreme biocompatibility [10]. Recently, thanks to the different association between synthetic and natural compounds, semi-synthetic hydrogels have been developed: this combines a good control of the structure of the scaffold and an optimal cellular response, thanks to the presence of natural components [23,24].

Smart Hydrogels
Smart hydrogels are polymers that can change their structural characteristics in response to different types of stimuli [25] and, once the latter has ceased, they are able to return to their equilibrium conditions [26]. These characteristics make this new generation of hydrogels more similar to the extracellular matrix; moreover, the increased controlled response make them suitable for the release of drugs only under certain conditions [10].
Depending on the stimulus, hydrogels can be distinguished into: -Chemical responsive: pH and oxidant status changes can alter hydrogel conformation.
However, pH changes cannot easily penetrate through materials.
-Physical responsive hydrogel: pressure, temperature, light induce changes in the physical state of hydrogels.
Hydrogels responsive to physical stimuli seem to be the most promising for medical applications, due to the relative ease of handling the physical stimulus. Thermosensisitve hydrogel are the most commonly used in recent years: below a certain temperature in fact, they are in the gel phase, otherwise they are present in liquid solution. Consequently, hydrogels at room and body temperature are ideal for modification in gel state after injection [10,15]. The chemical degradation rate of hydrogels can be adjusted by controlling the density of bound networks and enzymatic hydrolysis induced by proteases [15].

Nanogel and Microgel
Hydrogel of microscopic (microgel) and nanoscopic (nanogel) size were created, consisting of polymers of nanoparticles swollen in water that form three-dimensional networks of polymeric chains [10,28]. Their small size favours an increased available surface and, consequently, the ability to transport molecules [29]. In addition, the small size allows smart hydrogels to respond more quickly to environmental stimuli, as well as to reach inaccessible body locations.
However, nanogels induce a faster and more difficult to control release of drugs than microgels [10].

Hydrogel degradation.
The physical degradation of hydrogels provides space for cell migration and vascular infiltration in order to regenerate the suffering tissue [15,30]. The rate of degradation of hydrogels should therefore match with those of tissue regeneration in order to maintain the integrity of the new tissue and mechanical stability. The degradability of hydrogels depends on different physical factors such as the properties of the materials that contain them as well as different environmental factors [15,31].
The possibility of modulation the degradation rate of hydrogels has been also considered in the treatment of wounds with a poor tendency to healing [33]: no removal costs are required, with risk of suffering and, in the case of injuries, bleeding [34,35]. A hydrogel that degrades in coincidence with the wound repair process is able to significantly reduce the discomfort to the patient [31].

Chemoterapics, target therapies, immune checkpoint inhibitors
Recently, personalizing treatments with anti-cancer target therapies and immune therapies resulted in survival improvement but cardiovascular adverse effects experienced by an important percentage of patients [36][37][38]. Regenerative medicine is currently investigating advancements in drug delivery to targets, in order to avoid off-target effects, and strategies, such as cardioprotectants administration where delivery is not an option [39][40][41]. In particular, cardiotoxocities mainly arise from standard chemoterapics and targeted therapies. Immune checkpoint inhibitors and CAR T (chimeric antigen receptor T) cell therapies consitute the broad group of immunotherapies (table 1) [42]. They tend to cause rarer, but more fatal, cardiovascular events [43,44].  [45,46]. Arrythmias generation is a rising adverse effect from conventional therapy, such as alkylating agents, microtubule binding agents and immunomodulatory drugs. Target therapies also have a role in arrhythmia generation: mTOR Immune checkpoint inhibitors and CAR T cell therapies mainly cause fatal myocarditis, Takotsubo syndrome, acute cardiovascular syndromes and perciardial diseases. Mainly diagnosed by electrocardiography and echocardiography, these entities represent an important burden for oncological patients, requiring therapy suspension and invasive diagnostic/therapeutical strategies of management.

Anthracycline-induced cardiac diseases
Doxorubicin, an antineoplastic anthracycline antibiotic [51], was first discovered to cause heart failure in 1970s [52]. Adryamicin-treated cancer patients were evaluated during serial followups. Diagnostic electrocardiograms (ECGs) revealed significant QRS voltage decrease while echocardiographies revealed ventricular systolic failure, atrial dilation and other paramteric changes in 44 (11%) of total 399 patients [52]. Refractoriness to inotropic drugs and mechanical ventricular assistance was also clinically reported.
Left ventricular dysfunction is the most common cardiac side effect caused by doxorubicin.
Alteration of topoisomerase IIb and reactive oxygen species generation are some of the responsible mechanisms. One of the risks associated with drug exposure is the late development of cardiac disfunction. A potential 20% of patients is estimated to be diagnosed after chemotherapy completion [53][54][55].
The major risk factors for cardiotoxicity development are cumulative high doses, old age or young age, concomitant administration of other cardiotoxic drugs, radiation therapy exposure and cardiovascular diseases [56,57].

Alkylating agents and selected target therapies
Administration of cyclophosphamide, an antineoplastic agent used in combination with stem cell transplant, has been associated to reduced cardiac function, pericardial effusion and decreased ECG voltage [58][59][60]. It also causes hemorragic myopericarditis and complications, such as pericardial effusion.
Trastuzumab instead is broadly identified as a target agent, used for breast cancer and stomach cancer being it a monoclonal antibody against HER2 [61,62]. Adverse effects involving cardiac tissue from trastuzumab are mainly represented by cardiac dysfunction and alteration of echocardiographic parameters [63,64].
A preclinical study demonstrated that HER-2 is expressed in myocardiocytes of embryonic wild-type mice with a distribution pattern that is mostly restricted to the T-tubular network [65].
Instead, conditional mutant mice with cardiac-restricted HER-2 deletion showed dilated cardiomyopathy development after birth [65]. Although the role of HER-2 has not been completely unrevealed, several studies demonstrated that it is involved in cardiac hypertrophy as compensatory mechanism [65].

Strategies for specific drug delivery
Several delivery approaches have been developed for tumor targeting in preclinical studies.
Doxorubicin is now preferentially administered as pegylated liposomal doxorubicin to patients suffering from ovarian cancer, multiple myeloma, Kaposi sarcoma and others. It has been repeatedly investigated for specific tumor targeting in conjugation with hyper branched polymers or metronomic nano delivery. Results from clinical studies showed significant reductions in tumor size [66,67].
Anthracyclines have represented an important cause of morbidity and mortality in cancer survivors by causing cardiomyopathy, heart failure and even cardiac death. Reponsible mechanisms were identified in oxidative stress generation, defective mitochondrial biogenesis and consequent myofibrillar disarray and vacuolization [68]. Rising cardiac troponins T levels are expected in cardiotoxicity cases. Recently, the use of exosomes in conjugation to doxorubicin demonstrated to reduce the drug cardiac concentrations by 40% [69].
Adeno associated viruses were also tested for delivery of bevacizumab and other anti-VEGF antibodies. Bevacizumab was particularly tested for glioblastoma multiforme in preclinical studies [70]. Hicks  signifiy an efficient drug passage through the blood-brain barrier [70].
Anti-VEGF A antibodies were also expoilted in combination with adenoviruses. Patients in metastatic setting, suffering from lung cancer, were administered with AAV (adeno-associated virus) vector AAVrh.10alphaVEGF expressing an anti-VEGF A antibody equivalent to bevacizumab [71]. The adenoviral vector encoded the light chain and heavy chain complementary DNAs of monoclonal antibody A.4.6.1, a murine antibody that specifically recognizes human VEGF-A with the same antigen-binding site as bevacizumab [71].
After intrapleural administration, authors demonstrated a significant suppression of tumor growth, a decrease in the number of blood vessels and mitotic figures and increased animal survival [71].

Trastuzumab therapy
Cardiotoxicities from trastuzumab and reduced penetration of the monoclonal antibodies in tumors has led investigators to develop strategies of tecnical improvements. The tumor microenvironment is in fact composed by extraceullular matrix (ECM) and high interstitial fluid pressure (IFP) [72,73]. Interestingly, intravenous injection of trastuzumab has registered low therapeutical concentrations in the target, i.e., less than 0.01% of the dose per gram of tumor tissue [74,75]. The co-administration of antibodies and collagenase or hyaluronidase, given that collagen-I is the main component of ECM, is under investigation.
Recently, strategies concerning hylauronidase-incorporated hyaluronic acid-tyramine hydrogels and co-delivery of collagenase has therefore been tested for trastuzumab release. The weeks. Specifically, authors found a reduced tumour size when testing the combination in vivo [78].

ICI administration via hydrogels delivery systems
Immune checkpoint inhibitors have been also tested for specific target delivery via hydrogel formulations. Specifically, injectable polypeptide hydrogels have been tested for delivery of vaccines and checkpoint inhibitors [79]. Therapeutical agents (anti-CTLA-4/PD-1 antibodies) were mixed with tumor cell lysates and GM-CSF (granulocyte-macrophage colony stimulating factor) and tested upon the activity of dendritic cells (DCs) [79]. Results showed a strong Tcell reaction in vivo. Instead, when the hydrogel was coupled with antitumoral vaccines, IgGs production was also upregulated and IFN-γ, IL-4, and TNF-α levels were increased too [79].
Authors discovered that the hydrogel-based immunotherapy increased CD8 + T cells expression within the spleens and within tumors of vaccinated mice. Also, they were able to reduce the ratio of Tregs.
The research group by Han et al [80] developed a hydrogel for local therapy delivery by T mixing N1-(4-boronobenzyl)-N3-(4-boronophenyl)-N1, N1, N3, N3-tetramethylpropane-1,3diaminium (TSPBA) with poly (vinyl alcohol) (PVA) [80]. In another work, an anti-PD1 and Zebularine, a cytidine analog recently studied for chemoprevention, were delivered by an in situ formed hydrogel [81]. An enhanced immunotherapeutical response has also been achieved by the development and testing of a reactive oxygen species (ROS)-responsive polypeptide gel for facilitation of immune active drugs delivery. Specifically, it was tested in combination with an anti-PDL1 antibody and dextro-1-methyl tryptophan, inhibitor of indoleamine-2,3dioxygenase and facilitator of T cell proliferation arrest [82]. Yu et al [82] effectively demonstrated a reduction in local ROS levels and an in vivo increased response to melanoma.
Hydrogels have also been studied in combination with immune checkpoints-blocking DNA aptamers. They show advantages over antibodies in thermal stability and immunogenicity [83][84][85]. DNA aptamer hydrogels can be generated through rolling circle amplification (RCA), a tecnique that amplifies and hybridizes template sequences [85]. Results showed in vivo retention of the hydrogel at injection site: two groups were analyzed, a free PD-1 aptamer  PDS a has been shown to be a biomaterial capable of interacting with the elastic properties of the pulmonary artery. Furthermore, a synergy was found between the elastic properties of the PDS and the degradation time that was required once implanted, obviously in accordance with the application for which it was intended. It has been shown to be an ideal polymer with the following biological and mechanical properties [94,95]. As for the biological properties, firstly the PDS once in contact with the morphological structure of the pulmonary artery did not evoke excessive toxic or inflammatory responses of ECM. Second, it was metabolized, after having completed its favorable strengthening and remodeling action, without leaving a trace. Third, the PDS was easily processable [86][87][88][89]. Figure 3. The mechanical properties of the PDS were evaluated according to the application of the material, which was to contain the expansion of the pulmonary artery when it was implanted under systemic pressure, so that the polyester load was automatically distributed on the pulmonary artery tissue as it was degraded, thus reducing the effect of stress shielding [88,95].

Specific characteristic of Polydioxanone
Polydioxanone (PDS) has about 55% crystallinity and a glass transition temperature that varies between -10° and 0° C. It features a regular repetition of fundamental units that come together in chains folded into dense regions called crystallites. These join together by means of cross links giving the polymer a high tensile strength and a very high elastic modulus if compared to that of the amorphous analogue. These features are related to the degree of compactness of the polymer. It should be noted that no polymer can be organized into a total crystalline structure, so however high crystallinity may be found inside, partially amorphous regions will always be detectable. PDS is a very viscous, high-medium molecular weight polymer that has a slower biodegradation than those with lower molecular weight and lower viscosity. Notably, temperature plays a fundamental role with regard to material properties [88,89,94,95].

Biodegradation molecular mechanisms
PDS has been degraded primarily due to the loss of molecular weight and the loss of resistance of the material through an initial phase in which the degradation is due to a process of a chemical nature. The biological process and the total removal of the material take place later [86][87][88][89].
In detail, the polydioxanone polymer was degraded in two ways: dissolution and chain cleavage. In the case of dissolution, since PDs has hydrophilic domains, it was dissolved during normal physiological processes when solvent molecules, such as H2O present in pulmonary artery tissue, were absorbed into the polymer and were small enough to occupy the space between the chains of the macromolecules. The H2O molecules penetrated into the polymer acted as plasticizers, thus making the material more ductile as they reduced the number of secondary bonds between the chains. Furthermore, it is possible that this process could cause an altered crystallinity of the polymer. Both mechanical and thermal properties (e.g., glass transition temperature) can be affected by the absorption of solvent molecules. It was also possible that in extreme cases, the chains were soluble enough that few covalent bonds remained between the chains and the polymer that dissolved completely (figure 3 C) [89,[91][92][93][94].
Instead, the chain splitting involved the breaking of the primary bonds, rather than the secondary ones. There is a separation between the chain segments at the breaking point of the bond, which has led to a reduction in the molecular weight, which can have, as in the case of dissolution, significant consequences on the mechanical and thermal properties. Chain splitting can occur hydrolytically or by oxidation [94,95].
As regards hydrolysis, the H2O molecules have penetrated into the implanted material causing the splitting of the molecular bonds between monomers. This condition led to the splitting of the polymer chains into shorter chains. The main factors that produced the extension of PDS hydrolysis were: the reactivity of the functional groups of the main part of the polymer; the extension of the inter-chain links; the increase of the H2O available for the polymer. We have recorded in the vessel wall segments of non-degraded PDS due to probable reduction of the tissue H2O content but we cannot establish whether it is secondary to dissolution or hydrolytic cleavage [87-89, 94,95].
In the pulmonary artery wall, PDS was also eliminated by oxidation through the formation of free radicals that attack and break the covalent bonds that hold the chains together. The main factors influencing oxidation are mostly the reactivity of the functional groups of the main part of the polymer; the extension of the inter-chain links [89,[92][93][94].  [86-89, 94,95].

The use of Polydioxanone as crosslinked prosthetics: when and how
The pulmonary valve and trunk or the pulmonary autograft (PA i. e) may be used to treat aortic valve disease, however the pulmonary conduit is exposed to progressive expansion when it is implanted under the systemic pressure regimen. This operation was firstly executed by Donald Ross in 1967 and for this reason it is named Ross procedure (figure 3 A, B) [96].
The checks performed by our clinical teams have confirmed a trend towards dilatation of pulmonary autograft after the Ross procedure leading for a potentially risk of reoperation. The use of dacron as an external reinforcement is a choice adopted by many surgeons, however we have never been in favor of adopting it. We preferred to maintain the characteristics of the PA as living tissue while trying to induce remodeling of the pulmonary conduit [97][98][99][100][101][102].
The dacron, for various reasons, can not be considered suitable material for this reinforcement and the late outcomes of the use of this synthetic materials are increasingly revealing its limitations at both the clinical and biological level. Infact the dacron used by many surgeons in many institutions is unable to growth and fail to match the demanding need of a growing structure such as the pulmonary autograft especially when it is implanted in the pediatric population. Secondly, vascular compliance is poor and it has been estimated that Dacron has a stiffness that is 24 times greater than native aorta [86][87][88]95]. This would imply the loss of On the contrary pathological analysis performed after the use of resorbable polydioxanone to reinforce pulmonary artery revealed a partial resorption of the polyester, recording a preserved endothelial lining. This evidence suggested to confirm the absence of intimal tearing under pressure load. In addition, a medial thickening with thoroughly arranged fibromuscular cells mixed to abundant neo-formed connective tissue was identified (figure 3 D). Of note, using the polydioxanone the MMP-9 was detected to be overexpressed once the polyester was integrated into the ECM so to point out an ongoing matrix remodeling process [88,94]. The collagen architecture appeared well organized with a compact density and distribution in the elastic region of the pulmonary artery. Taken together, these results concretely suggest a shift towards a process of elastic remodeling and neo-arterialization. In confirmation of these findings, it was possible to note the presence of a scarce inflammatory infiltrate with rare macrophages or monocytes that colonized the reinforced arterial wall. Pathoanatomical evidence reliably suggested that the biomaterial did not elicit an exuberant foreign body inflammatory reaction over time [87][88][89]91,92,94].
We induced a neoarterialization of the PA which was placed in the aortic position and therefore

Conclusions
Hydrogel formulations have recently been regarded as novel delivery opportunities. Despite this, they have been studied for long and are conquering vast clinical fields. Anticancer therapies are one of the best applications, due to their severe side effects including cardiovascular toxicities, namely heart failure and myocarditis. After preclinical studies implementations, trials will be held, due to the extraordinary results obtained from in vitro and animal in vivo experimentations.