Methodology and work schedule
The tasks and stages to be developed for each of the studies to obtain biodegradable polymers impregnated with pharmacoactive substances through high-pressure processes and the tasks common to both are listed below.
Task I. Extraction and characterization of mango leaf extracts
Mango leaf extracts have already been studied by this Research Group in previous projects, so the procedure, optimal operating conditions and chemical analysis and some of the functional analyses are already known. However, it is necessary to include new functional tests with regard to their use in biomedical implants, since no references have been found on the use of mango leaf extract in scaffolds and stents.
Task I.1. Obtaining the mango leaf extracts
The extracts will be obtained from leaves of Mango indica L, variety Kent, provided by the Experimental Station “La Mayora”, of the Superior Council of Scientific Research with which some members in the research team already collaborate. The equipment and operating conditions described in previous works will be applied .
Task I.2. Characterization of the extract
Activity I.2.1. Determining the extract’s antimicrobial capacity: using Escherichia coli and Staphylococcus aureus as models of Gram- and Gram+ bacteria, respectively.
Activity I.2.2. Determining the extract’s anti-inflammatory capacity: efficacy against denaturalization inhibition of egg albumin by thermal action following the protocols described in the literature. This test has not been previously conducted by the research team and, therefore, some adjustments will be necessary.
Activity I.2.3. Determining cellular proliferation in the extract: endothelial colony-forming cells (ECFCS) will be cultivated and incubated in extracts at different concentrations. Subsequently, the levels of the protein KI67, a marker of nuclear proliferation, will be analyzed by means of immunohistochemical assays in order to evaluate the levels of KI67 under the different culture conditions.
No difficulties to carry out this task have been contemplated. However, as a contingency plan, the use of leaf extracts from other agricultural by-products such as leaves from olive tree, red grape skin or other anonaceous plants that have already been used by the Group in previous works is proposed.
Task II. Study of the foaming process.
The operating procedure as well as the operating conditions (different polymer mixtures at different ratios, type of contact, compression-decompression rates, pressure, temperature and CO2 flow, etc.) will be optimized.
Task II.1. Selection of the conjugated polymer and the non-conductive polymer
Activity II.1.1. The study will be started using polyalinine and polypyrrol, as conjugated polymers and PLA and PCL as non-conductive polymers. The foaming process of both types of polymers will be studied separately. A RESS equipment will be employed as follows: insertion of the extract into the polymer solubilization cell, injection of the CO2 up to the specified pressure and temperature conditions (between 100-350 bar, and between 35-65ºC). In addition, the agitation speed will be evaluated in the range between 0 and 80 rpm. After keeping them in contact long enough so as to assume that equilibrium has been reached, the system will be depressurized at varying depressurization rates with the range 1 to 100 bar/min in order to directly and visually evaluate the eventual foaming of the combined polymer.
Activity II.1.2. Hydrothermal treatment will be applied to both the non-processed and processed polymers to verify how their physical properties have been modified/improved for impregnation and/or end use. For this purpose, a 300 mL Autoclave Engineers equipment fitted with mechanical agitation will be used under different operating conditions for pre-treatment (polymers) or post-treatment (foaming). The operating temperature will vary between 50 and 120ºC in order to determine the optimal temperature level, which will depend on the polymer vitreous transition and melting temperature. Other parameters such as slow heating/quick cooling; short reaction times: 1-15 min; pressure —to maintain water as a liquid or vapor phase
—; sample placement — submerged in the liquid or in the upper space (steam) — will also be optimized.
These preliminary studies will allow us to determine the optimal operating conditions for the subsequent tasks.
Task II.2. Selecting the mixture of conjugate polymer and non-conductive polymer Activity II.2.1. Once the best operating conditions for the foaming of the four polymers based on their porosity and for their use as scaffolds or stents have been determined, the optimal mixture will also be determined. Different ratios will be tested and the equipment will operated in the same way as for Activity I.1.1.
Activity II.2.2. Hydrothermal treatment will be applied to both untreated polymer mixtures and treated combined polymers to verify whether their physical properties are improved/modified when the same procedure as in Activity II.1.2 is applied to them.
If the expected results are not obtained in this task, other polymers, such as poly (3,4- ethylenedioxythiophene) (PEDOT) in the case of the conjugated polymer and PLGA as a non-
conductive polymer, as well as all their possible combinations would be tested. Should the expected results still not be obtained with the new combined polymers, other options will be contemplated according to previous studies involving the use of scCO2. Moreover, when necessary, any other available high pressure equipment will be employed for the tests until satisfactory results are achieved.
Task III. Study of the impregnation process
Like for Task II, the operating procedure and the operating conditions are to be optimized. Task III.1. Study of the contact method between the combined polymer and the extract Activity III.1.1. The previously formed polymer together with the extract are inserted into the impregnation cell, which is equipped with an agitation system. The cell is pressurized with the CO2 under the impregnation conditions, and the agitation system is allowed to run for as long as necessary for the impregnation process of the polymer to complete.
Activity III.1.2. The previously formed combined polymer is introduced into the impregnation cell on its own. The cell is pressurized with a saturated mixture of CO2-sc and extract according to the established impregnation conditions and the system is allowed to agitate for as long as necessary for the polymer impregnation process to take place.
Task III.2. Study of the contact method between the polymer and the bioactive substances in a single step.
This task consists on developing a single step method that brings the previously collected bioactive substance and the polymer into contact with each other.
Activity III.2.1. Impregnation and foaming in a single step, where the extract and the polymer are placed in the same container and operate in batch mode.
Activity III.2.2. Impregnation and foaming in a single step, where the polymer is introduced into the container and the extract is dissolved in CO2-sc. Semi-continuous mode.
The contact method that provides the greatest impregnation and pore uniformity will be selected. All the experiments will be carried out in an Iberfluid SSI impregnation equipment. The variables to be studied are the same as in Task II.1. In addition, the addition of co-solvent will also be evaluated and the impregnation time will be determined between 1 and 24 hours. The experiments will be specifically designed so that these parameters can be determined.
Since several impregnation methods are being analyzed, when one of the impregnation methods does not lead to successful results, any study efforts will be focused on the other methods that may lead to optimize the impregnation results.
Task IV. Characterization of the products obtained
Task IV.1. Mechanical properties.
Tensile and compressive strength will be measured by means of a MTS Criterion C45 mechanical press.
Task IV.2. Textural properties.
Specific surface, pore volume and pore size will be measured using Micromeritics ASAP 2420 at 77 K. The specific area will be calculated by the BET method and the microporosity by the t-plot method.
Task IV.3. Physical-chemical properties.
Electrical properties (impedance spectroscope model Solartron SI 1296), FTIR analysis (Bruker Tensor 37 infrared spectrometer), surface chemical composition by XPS (Kratos Axis Ultra DLD), thermogravimetric analysis (TGA 50 from TA Instruments), scanning electron microscopy (Nova NanoSEMTM 450) to evaluate the structure of the formed cells and the distribution of the pharmacoactive particles within the impregnated polymers.
Task IV.4. Functional properties.
The same properties described in Activity I.2 will be evaluated after adapting them for the study of the impregnated polymer. The main inconvenience is that the measurement by confocal microscopy presents some difficulties when cell growth on an opaque polymer is to be determined.
Task V. Study of the release kinetics of the impregnated substances
Task V.1. Acquisition of the release profiles
The release kinetics of the impregnated bioactive substances will be determined by means of an HPLC/UPLC chromatograph that would monitor the concentration levels of several substances at the same time.
Task V.2. Modelling of the release process
The modified Higuchi model will be applied to describe the release of the compounds from the porous matrices. The relaxation time of the polymer-solvent system will be compared to the characteristic diffusion time of the solvent. The comparative analysis of these times will allow to determine if the process is controlled by the diffusion stage and if the release of the drug is mainly determined either by Fick’s law or by the swelling phenomenon stage.
If this model does not provide satisfactory results, other models from the literature, such as Korsmeyer-Peppas or Peppas-Sahlin, will be considered.
Task VI. Study of the functionality of the products obtained from in vitro tests and analysis of the viability of the impregnated polymer for clinical practice
Task VI.1. Toxicity, proliferation and apoptosis assays on human umbilical cord endothelial colony forming cells (HUVECs).
The effect of incubating human umbilical cord endothelial colony forming cells (HUVECs) with the impregnated materials will be evaluated. The effect of these cells with the impregnated materials on cell viability (with the MTT reagent), proliferation (by flow cytometry with the Click- IT EDU reagent and/or by immunocytochemistry evaluating the levels of KI67), and apoptosis (by flow cytometry measuring the levels of Annex V and propidium iodide by flow cytometry) will be evaluated.
Task VI.2. Adhesion and proliferation tests.
Colony-forming progenitor endothelial cells (CFE’s) will be plated in order to evaluate the degree of adhesion and proliferation of these cells.
Task VI.3. Assessment of the viability of the resulting impregnated polymers for their use in bioabsorbable implants with pharmacological properties.
Given that this project concerns Challenge 1 regarding Health, Demographic Change and Welfare, a clinical analysis of the viability of the materials for their use in bio-absorbable implants is considered to be of great importance. In order to carry out this analysis it will be necessary to have available all the data compiled from the remaining activities in Task VI. The Research Team has incorporated a researcher from the clinical area who is a specialist in cardiology and the use of bio-absorbable polymeric pharmacological implants and the Task Team has also incorporated two Doctors in Molecular Biology for the in vitro assays.