WO2021005536A1 - Substrat cellulaire ou milieu de culture pour induire une réponse cellulaire, procédés et utilisations de ceux-ci - Google Patents

Substrat cellulaire ou milieu de culture pour induire une réponse cellulaire, procédés et utilisations de ceux-ci Download PDF

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WO2021005536A1
WO2021005536A1 PCT/IB2020/056434 IB2020056434W WO2021005536A1 WO 2021005536 A1 WO2021005536 A1 WO 2021005536A1 IB 2020056434 W IB2020056434 W IB 2020056434W WO 2021005536 A1 WO2021005536 A1 WO 2021005536A1
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growth factor
biomaterial
acrylamide
molecularly imprinted
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Simão Pedro BARBOSA TEIXEIRA
Rui Miguel DE ANDRADE DOMINGUES
Maria Manuela ESTIMA GOMES
Rui Luís GONÇALVES DOS REIS
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Association For The Advancement Of Tissue Engineering And Cell Based Technologies & Therapies (A4Tec) - Associação
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/268—Polymers created by use of a template, e.g. molecularly imprinted polymers
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2531/00—Microcarriers

Definitions

  • the present disclosure describes the use of molecularly imprinted polymer nano- or microparticles (MIPs) as selective ligands for biomacromolecules, namely bioactive proteins, combined with a suitable cell substrate or culture medium, in particular for stem cell culture.
  • M IP-decorated substrates selectively bind and sequester the imprinted bioactive proteins of exogenous or endogenous origin, amplifying and inducing specific stem cell responses, such as survival, proliferation, and differentiation.
  • the present disclosure is useful tissue engineering and regenerative medicine.
  • GFs growth factors
  • stem cells constitute the golden triad of tissue engineering approaches.
  • Growth factors are bioactive proteins secreted by cells. GFs are responsible for eliciting a number of biological responses such as cell survival, cell proliferation, cell growth, and cell differentiation. Thanks to these properties, GFs have for long been identified as potent therapeutic agents and tested in various clinical trials.
  • a number of limitations has hindered these molecules from realizing their full potential, namely their prohibitive costs, low shelf-life, and fast degradation after administration. Due to these stability limitations, administration of repeated supraphysiologic doses are usually required to achieve therapeutic efficacy. This often leads to serious undesired off-target side-effects and costly treatments.
  • GFs are secreted by cells into the extracellular environment where they are bound by molecules in the extracellular matrix (ECM). In this manner, their local concentration and presentation to cell receptors is tightly regulated for optimal effects.
  • ECM extracellular matrix
  • Recent biomedical strategies have found inspiration in this phenomenon, developing GF sequestering systems that allow for controlled, precise, sustained, and localized action of these proteins. Some of these have made use of GF-binding components of the ECM, such as heparin, fibronectin or fibrinogen.
  • Document WO2014113573 describes hydrogel cell matrix systems for the support, growth, and differentiation of a stem cell or progenitor cell and methods for making such hydrogel cell matrices.
  • the system described in WO2014113573 makes use of heparin as GF controlled release moiety, but this binding is promiscuous, thereby not allowing the targeting of a specific GF for a particular effect.
  • Targeted ligands are also being researched for the capture of specific GFs. These include antibodies, aptamers, small molecules, and peptides. The latter 3 have small to moderate sizes and thus have good permeability, relatively low production costs, and relatively high ease of manipulation. But small molecules lack in specificity, while peptides and aptamers are sensitive to degradation and are rapidly cleared. Antibodies have high specificity and affinity, finding many applications in the biological field. However, they are also proteins and therefore present the same limitations as GFs: large size, low stability, high production costs, and potential immunogenicity.
  • Molecular imprinting is a process in which a monomer solution is mixed with a template - target molecule - establishing temporary interactions with it. Polymerization is then triggered and, after removing this template, the resulting molecularly imprinted polymer (MIP) is endowed with cavities with shape and molecular interactions complementary to it. Upon coming into contact with the template, the MIP is able to selectively rebind it in a reversible fashion which has earned MIPs the epithet of "plastic antibodies”.
  • MIPs answer to several of the previous ligand limitations, which led to the growth of their use in various fields, like separation, sensing, and targeted drug delivery. [0009]
  • the binding affinity of MIPs has increased over the years to levels similar to those of natural antibodies, which possess some of the most selective and avid non-covalent interactions in nature.
  • their synthetic polymeric origin means they possess much higher stability in terms of temperature, pH, and enzymatic action. The shelf life is longer, allowing storage of the product at room temperature for long periods of time (months to years), as well as easier long distance transportation by dispensing the need for a cold chain.
  • Another advantage is that they can be synthesized from a variety of monomers with different functional groups, allowing the use of different coupling chemistries to immobilize the particles to a surface or scaffold. This also allows a wide range of interactions to occur between monomers and template, meaning each polymer can be tweaked in terms of components to generate higher affinity and selectivity for the target.
  • the preparation procedure can be easily scaled up, allowing the production of large quantities at much inferior costs, when compared to biological alternatives.
  • MIPs are produced by having a mixture of monomers come into contact and establish transient interactions with the template. These can be covalent or non- covalent bonds. The mix is then polymerized, and the template removed, to produce imprinted cavities in the polymer matrix, as disclosed in W00130856 (Al). W00130856 (Al), however, claims the use of MIPs in solution as drugs, receptor-specific ligands, or components of pharmaceutical compositions or food, not in combination with biomaterials for purposes of biomolecule sequestration, stem cell fate modulation and tissue engineering.
  • Functional monomers can be one or more compounds, chosen to maximise interaction with the template and according to the specific needs for stability, biodegradability, biocompatibility, thermoresponsiveness, among others. These include acrylates, methacrylates, acrylamides, vynil monomers, allyl monomers, among others.
  • MIPs molecularly imprinted nano- or microparticles
  • This disclosure provides a solution based on MIPs for the precise control over stem cell fate, in terms of either expansion or differentiation, mitigating or potentially avoiding the need for addition of exogenous GFs in cell culture and in the fabrication of tissue engineered constructs.
  • This disclosure also provides a method for using MIPs as a component that simultaneously has higher stability, specificity, scalability and lower production costs than other sequestering moieties.
  • a method for the control of stem cell fate by sequestering specific biomacromolecules, particularly bioactive proteins, at a suitable biomaterial substrate for cell culture.
  • This method consists in using as selective ligands polymeric nanoparticles molecularly imprinted against a template molecule (MIPs).
  • MIPs template molecule
  • the proposed molecularly imprinted nano- or microparticles may considerably reduce the costs and improve efficacy of cell expansion protocols, production of phenotypically defined engineered tissues, and/or their engraftment.
  • said template can be a growth factor.
  • EGF epidermal growth factor
  • PDGF platelet-derived growth factor
  • TGF transforming growth factors
  • FGF fibroblast growth factors
  • BMPs bone morphogenic proteins
  • GFs can be isolated from native tissues or produced by recombinant means in bacteria or yeast, with both being available commercially from vendors, such as Sigma, ThermoFisher, or PeproTech.
  • the template can be an epitope selected from an exposed portion of the target molecule. In the case of proteins like GFs, the epitope corresponds to a peptide, which can be linear or adopt a conformation similar to the portion of the native protein.
  • an epitope of transforming growth factor-bB was used as template instead of the whole protein.
  • a conformational epitope was designed to capture the superstructure of the exposed a-helix of TGF-bB at the N-terminus of the growth factor. This was achieved using apamin, an a-helix-containing disulfide-bridged peptide derived from bee venom, as a scaffold for designing a novel peptide that mimic the TGF-bB domain featuring similar structure.
  • synthetic polymer NPs were surface imprinted with a growth factor (GF) epitope, namely TGF-bB, as template molecule.
  • GF growth factor
  • TGF-bB growth factor-bB
  • Different combinations of monomers and cross-linker were used to produce a series of imprinted (MINP) and non- imprinted (NINP) polymer nanoparticles by inverse microemulsion polymerization.
  • MINP and NINP were characterized by different microscopy and spectroscopy techniques.
  • the affinity between the MINP formulations and TGF-bB was studied by western blot (WB) and surface plasmon resonance (SPR). WB and SPR revealed to be useful techniques for this purpose, and optimized experimental methods have been established to characterize the affinity between MINP and target molecule.
  • MINP/NINP formulations were incubated in PL and the composition of their protein corona was analysed after selected washing steps.
  • the intensity of the band corresponding to mature TGF-bB dimeric form is much higher in the hard corona of MINP6 than in NINP6, suggesting higher specificity of the imprinted formulation toward TGF-bB.
  • This effect was also observed for MINP/NINP1 although to a lower extent, suggesting that the chemical composition of MINP6 contributes more to the desired abiotic affinity between MINP and target protein.
  • TGF-bB The specificity of the imprinted recognition sites to TGF-bB was assessed by analysing the presence of other GF in NPs protein corona, namely TGF-bI and VEGF. Differently from the results obtained for TGF- bB, the presence of these two GFs on the hard corona of NPs was detected but without evidencing preferential binding by the imprinted vs non-imprinted formulations.
  • the affinity of the MINP/NINP to TGF-bB epitope was studied by SPR.
  • MINP1 and MINP6 formulations have shown higher affinity to the TGF-bB epitope than the respective NINP whereas the MINP2/NINP2 formulations have shown similar affinity, confirming the WB results.
  • MINP/NINP6 were immobilized on tissue culture coverslips.
  • the genetic profile and phenotypic commitment of human adipose-derived stem cells (hASCs) cultured on these substrates was evaluated.
  • hASCs human adipose-derived stem cells
  • MINP6-surfaces were able to promote the commitment of hASCs towards the chondrogenic lineage by cell modulating the autocrine and paracrine cell signaling due their ability to capture the cell secreted TGF-bB.
  • An aspect of the present disclosure relates to a molecularly imprinted biomaterial for inducing a cellular response comprising: a biocompatible non-degradable polymer made from monomers selected from a list consisting of: acrylates, methacrylates, acrylamides, vinyl or allyl monomers, or mixtures thereof;
  • molecularly imprinted biomaterial has a shape of an epitope of a biomacromolecule selected from the following list: a growth factor, cytokine, chemokine or combinations thereof.
  • a biocompatible polymer synthetized using monomers selected from acrylates, methacrylates, acrylamides, vynil monomers or allyl monomers wherein the molecularly imprinted biomaterial selectively recognizes and sequesters a biomacromolecule selected from the following list: a protein, a growth factor, cytokine, a chemokine, or combinations thereof; by the shape and chemical organization of the polymer precursor monomers.
  • the biocompatible non-degradable polymer is acrylamide.
  • the monomer and cross-linker are selected from a list consisting of: acrylamide, methacrylic acid, 2-aminoethyl methacrylate, N,N-methylene- bis-acrylamide, or combinations thereof.
  • the monomer and cross-linker combination is : acrylamide and N,N-methylene-bis-acrylamide, or
  • the molar ratio of acrylamide/ N,N-methylene-bis-acrylamide is from 7/1 to 10/2, preferably 8.18/1.
  • the molar ratio of acrylamide/ methacrylic acid is from 8/1 to 11/2, preferably 9/1.
  • the molar ratio of acrylamide/ 2-aminoethyl methacrylate is from 16/1 to 25/1, preferably 19/1.
  • the molecularly imprinted biomaterial has a shape of an epitope of a growth factor, preferably a growth factor selected from: epidermal growth factor, platelet-derived growth factor, transforming growth factor, fibroblast growth factor, or mixtures thereof, preferably transforming growth factor ⁇ .
  • the molecularly imprinted biomaterial further comprises a substrate, wherein the substrate is a mesh, a hydrogel, a scaffold, a fibre, or combinations thereof.
  • the biomaterial of the molecularly imprinted biomaterial is a microparticle or a nanoparticle, preferably a nanoparticle.
  • the substrate in the molecularly imprinted biomaterial is cell differentiation factor.
  • the induced cellular response is selected from cellular proliferation, cellular survival and/or cellular differentiation.
  • An aspect of the present disclosure relates to a method for producing the molecularly imprinting biomaterial by inverse microemulsion polymerization, comprising the following steps: adding an epitope to a dissolved monomer and a cross-linker to form a mixture; agitating the mixture;
  • An aspect of the present disclosure relates to a cell substrate or a culture medium for inducing a cellular response, comprising a molecularly imprinted biomaterial comprising: a biocompatible non-degradable polymer selected from acrylates, methacrylates, acrylamides, or mixtures thereof; wherein the molecularly imprinted biomaterial has a shape of an epitope of a biomacromolecule selected from the following list: a growth factor, cytokine, chemokine or combinations thereof.
  • the induced cellular response is selected from cellular proliferation, cellular survival and/or cellular differentiation.
  • the molecularly imprinted biomaterial has a shape of an epitope of a growth factor.
  • growth factor is selected from: epidermal growth factor, a platelet-derived growth factor, a transforming growth factor, fibroblast growth factor, or mixtures thereof, preferably transforming growth factor-bB.
  • the biocompatible polymer can be synthetized using monomers selected from acrylates, methacrylates, acrylamides, vinyl or allyl monomers, and a cross-linker.
  • the biocompatible non-degradable polymer can be acrylamide.
  • the monomer and cross-linker may be selected from a list consisting of: acrylamide, methacrylic acid, 2-aminoethyl methacrylate, N,N-methylene- bis-acrylamide, or combinations thereof.
  • the monomer and cross-linker combination may be acrylamide and N,N-methylene-bis-acrylamide or acrylamide, N,N-methylene-bis- acrylamide and methacrylic acid, acrylamide, N,N-methylene-bis-acrylamide and 2- aminoethyl methacrylate.
  • the molar ratio of acrylamide/ N,N-methylene-bis-acrylamide is from 7/1 to 10/2, preferably 8.18/1.
  • the molar ratio of acrylamide/ methacrylic acid is from 8/1 to 11/2, preferably 9/1.
  • the molar ratio of acrylamide/ 2-aminoethyl methacrylate is from 16/1 to 25/1, preferably 19/1.
  • the substrate can be a mesh, a hydrogel, a scaffold, a fibre, or combinations thereof.
  • the molecularly imprinted biomaterial can be a nanoparticle or a microparticle.
  • Another aspect of the present disclosure relates to the use of the molecularly imprinted biomaterial described in the present disclosure for scientific research, medical research, medical diagnostics, medical therapy purposes, tissue engineering and regenerative medicine.
  • Another aspect of the present disclosure relates to the use of the molecularly imprinted biomaterial described in described in the present disclosure for sequestering biomacromolecules and controlling cell differentiation.
  • Another aspect of the present disclosure relates to the use of the molecularly imprinted biomaterial described in the present disclosure as an inducer of the cellular response, preferably as cellular proliferation inducer, cellular survival inducer and/or cellular differentiation inducer.
  • Another aspect of the present disclosure relates to a method for producing the molecularly imprinting biomaterial further comprising following steps: washing the mixture with ethanol;
  • the monomer and cross-linker in the method for producing the molecularly imprinting biomaterial are selected from: acrylamide, methacrylic acid, 2-aminoethyl methacrylate, N,N-methylene-bis-acrylamide, or combinations thereof.
  • the surfactant in the method for producing the molecularly imprinting biomaterial is selected from: an anionic (AOT) surfactant, a non-ionic surfactant, or combinations thereof.
  • AOT anionic
  • the epitope in the method for producing the molecularly imprinting biomaterial is transforming growth factor b-3 (TGF -3) epitope.
  • the mixture in the method for producing the molecularly imprinting biomaterial is agitated for 30 minutes at a temperature from 18 °C to 40 °C, preferably 20-22 °C.
  • the dropping of the mixture into the deoxygenated hexane solution in the method for producing the molecularly imprinting biomaterial further comprise stirring the mixture for 1 hour under nitrogen atmospheric condition.
  • the adding of APS and TEMED in the method for producing the molecularly imprinting biomaterial further comprising mixing the mixture for 2 hours under vacuum protection.
  • the crosslinking agents in the method for producing the molecularly imprinting biomaterial are APS and TEMED.
  • Another aspect of the present disclosure relates to the use of the molecularly imprinted biomaterial for scientific research, medical research, medical diagnostics, medical therapy purposes, tissue engineering and regenerative medicine.
  • Another aspect of the present disclosure relates to the use of the molecularly imprinted biomaterial for sequestering biomacromolecules and controlling cell differentiation.
  • the present disclosure relates to the use of MINPs as ligands with abiotic affinity for GFs in alternative to their biological analogs in TERM applications.
  • the present disclosure also improves the affinity and selectivity of MINPs for growth factor (GF), in particular to the target TGF-bB and demonstrate their functionality as modulators of cell behavior, on both 2D surfaces and 3D cultures, opening the door to a myriad of applications.
  • GF sequestering biomaterials have previously shown to be able to control cell fate and promote effective tissue regeneration by capturing and retaining endogenous GFs, or by the delivery of ultra-low doses of recombinant GFs.
  • MINPs of the present disclosure produced with epitope templates can be used as selective ligands to capture a target GF and direct cell responses, dispensing expensive biomaterial functionalization or media supplementation. This holds the potential to significantly reduce the high costs associated with current tissue engineering products.
  • the cell substrate and culture medium of the present disclosure has a significant impact on cell biomanufacturing, where strategies to reduce GF usage are of particular importance.
  • the use of MINPs in cell production pipelines can not only make these processes more economic but also allow the decoupling of nutrient delivery (in media) from biological stimulation, making large scale expansion of cells more efficient.
  • the present disclosure contributes to bypass or minimize the limitations related with the use of labile, expensive, and animal-derived biomolecules in TERM applications, widening the adoption of molecular imprinting concepts in the biomedical field.
  • Figure 1A shows confirmation of the formation of water-in-oil emulsions stabilized by the amphiphilic epitope. From left to right - Mixed solvent (500 pL water and 500 pL hexane) with 1 mg brij-30; 1 mg epitope; control (no surfactant, no epitope).
  • Figure IB shows the Transmission Electron Microscopy (TEM) images of MINPs and
  • Figure 1C shows the Transmission Electron Microscopy (TEM) images of NINPs. MINP3 and NINP3 nanoparticles were used as representative examples.
  • Figure 2 shows the result of Real-time SPR analysis of the molecular imprinted and non-imprinted nanoparticle formulation affinity for TGF- 3 epitope. Plasmon angle variation as response to successive injections of increasing concentrations of MINP1 and NINP1 ( Figure 2A), MINP2 and NINP2 ( Figure 2B), and MINP6 and NINP6 ( Figure 2C).
  • Figure 3 shows the result of Western blot analysis to determine the presence of TGF- 3 among the protein corona of the non-imprinted and imprinted nanoparticles of the formulations NINP1 and MINP1, NINP2 and MINP2, NINP6, and MINP6 after incubation in a 25% PL solution.
  • Lanes from left to right molecular weight ladder; soft protein corona (C); hard protein corona after 3, 6 and 10 washing steps.
  • C soft protein corona
  • hard protein corona after 3, 6 and 10 washing steps.
  • Figure 4 shows the result of Western blot analysis to determine the presence of TGF-bI among the protein corona of NINP1/MINP1 and NINP6/MINP6 formulations after incubation in a 25% PL solution. Lanes from left to right: molecular weight ladder; soft protein corona (C); hard protein corona after 3, 6 and 10 washing steps.
  • Figure 5 shows the result of Western blot analysis to determine the presence of VEGF among the protein corona of NINP1/MINP1 and NINP6/MINP6 after incubation in a 25% PL solution. Lanes from left to right: molecular weight ladder; soft protein corona of (C); hard protein corona of after 3, 6 and 10 washing steps.
  • Figure 6 shows the result of the Dot blot analysis to determine the presence of rhTGF-bB in the protein corona of NINP6/MINP6.
  • Figure 6a is a representative picture of observed blot;
  • Figure 6b shows the mass of remaining GF adsorbed after either 3, 6, or 10 washes, per mg of particles (** p ⁇ 0.01, statistical analysis by two-way ANOVA followed by Sidak multiple comparison).
  • Figure 7 shows the AFM images of tissue culture coverslips coated with polydopamine (PDA) and the subsequent MINP6 and NINP6 functionalized surfaces.
  • PDA polydopamine
  • Figure 8 shows the fluorescence microscopy images of tissue culture coverslips functionalized with either MINP6 or NINP6 after treatment with rhTGF- 3, followed by immunolabelling for this protein.
  • Figure 9 illustrates the qPCR quantification of expression of genes of interest. Highlighted are genes with significant differences in expression between cells cultured on MINPs and NINPs.
  • Figure 10 shows the fluorescence images of immunocytochemistry assay on hASCs seeded on MINP6- or NINP6-functionalised surfaces.
  • Blue represents nuclei stained with DAPI; red represents the cytoskeleton stained with phalloidin; green represents proteins of interest marked by immunostaining (either SOX9 or SCX).
  • Figure 11 shows the quantification of SCX and SOX9 protein expression levels from immunostaining fluorescent images.
  • FIG. 12 Biological effects of NINP6 supplementation in hASCs cultured as 3D pellets a - Schematic illustration of experimental procedure and results. hASC pellets cultured with NINP6 showed increased matrix production compared to those cultured with NINP6.
  • b Histological analysis of hASC pellets. MT - Masson trichrome (light microscopy; scale bar: 150 pm). AB - Alcian blue (light microscopy; scale bar: 150 pm). Col II - collagen II immunohistochemistry (confocal microscopy; scale bar: 100 pm) c - Section area of pellets measured from MT and AB-stained samples (**** p ⁇ 0.0001). d - Composition of pellets cultured with MINP6 or NINP6 (percentage of cells vs. matrix)
  • the present disclosure describes the use of molecularly imprinted nano- or microparticles (MIPs) as selective ligands for biomacromolecules, namely bioactive proteins combined with a suitable substrate for stem cell culture.
  • M IP-decorated substrates selectively bind and sequester the imprinted bioactive proteins of exogenous or endogenous origin, amplifying and inducing specific stem cell responses, such as survival, proliferation, and differentiation.
  • the present disclosure is useful in tissue engineering and regenerative medicine.
  • the physico-chemical properties of molecularly imprinted polymer nanoparticles (MINP) and molecularly non-imprinted polymer nanoparticles (NINP) were characterized.
  • Figure la shows confirmation of the formation of water-in-oil emulsions stabilized by the amphiphilic epitope. From left to right - Mixed solvent (500 pL water and 500 pL hexane) with 1 mg brij-30; 1 mg epitope; control (no surfactant, no epitope).
  • Figure lb shows the transmission electron microscopy (TEM) images of MINPs and
  • Figure lc shows the images for NINPs. MINP3 and NINP3 nanoparticles were used as representative examples.
  • Figure 2 shows the results of Real-time Surface Plasmon Resonance (SPR) analysis of the molecularly imprinted and non-imprinted nanoparticle formulation affinity to TGF ⁇ 3 epitope.
  • Figure 2a shows the plasmon angle variation as a response to successive injections of increasing concentrations of MINP1 and NINP1
  • Figure 2b shows the plasmon angle variation as a response to successive injections of increasing concentrations of MINP2 and NINP2
  • Figure 2c for MINP6 and NINP6.
  • MINP1 and MINP6 formulations showed higher affinity for TGF ⁇ 3 epitope than their respective NINP.
  • MINP2/NINP2 formulations both showed similar affinity ( Figure 2), confirming the western blot results.
  • the platelet lysate (PL) is a source of cytokines with therapeutic interest including growth factors (GFs) such as PDGF, VEGF, FGF, IGF-1, EGF, TGF-b among others.
  • GFs growth factors
  • the GFs of TGF-b family are amongst the most representative GFs in PL with an overall concentration of 125 ⁇ 15 ng/mL.
  • the ability of the MINP to selectively isolate TGF ⁇ 3 from PL and retain this GF after several washes was assessed by western blot, as shown in Figure 3.
  • Figure 3 shows the result of the Western blot analysis to determine the presence of TGF ⁇ 3 among the protein corona of the non-imprinted and imprinted nanoparticles of formulations NINP1 and MINP1, NINP2 and MINP2, NINP6, and MINP6 after incubation in a 25% PL solution.
  • Lanes from left to right molecular weight ladder; soft protein corona (C); hard protein corona after 3, 6 and 10 washing steps.
  • C soft protein corona
  • hard protein corona after 3, 6 and 10 washing steps.
  • NINP2 and MINP 2 formulations only the soft protein corona and the hard protein corona after 1 and 3 washing steps are presented.
  • the albumin band (around 55 kDa) and several bands consistent with the large latent complex (120- 160 kDa), immature (49 kDa) and mature dimeric (24 kDa) forms of TGF-bB were clearly identifiable.
  • the contribution of each TGF-bB form to the soft protein corona depends on the nanoparticles formulation. For instance, NINP2 and MINP2 formulations have stronger bands at 49 kDa, 44 kDa, and 34 kDa.
  • NINP1, MINP1, NINP6 and MINP6 formulations depict a similar composition with TGF-bB positive bands at >120 kDa, 49 kDa and 24 kDa.
  • the specificity of the recognition sites imprinted on to TGF-bB was also assessed.
  • the presence of other GFs within nanoparticles protein corona either from TGF-b family, namely the isoform TGF-bI, or from other GFs families (VEGF) was analysed.
  • Figure 4 depicts the western blot analysis to determine the presence of TGF-bI in the NINP1/MINP1 and NINP6/MINP6 protein corona.
  • Figure 4 shows the result of the Western blot analysis to determine the presence of TGF-bI among the protein corona of NINP1/MINP1 and NINP6/MINP6 formulations after incubation in a 25% PL solution. Lanes from left to right: molecular weight ladder; soft protein corona (C); hard protein corona after 3, 6 and 10 washing steps. [00103] It can be observed from Figure 4, the 48 kDa TGF- b ⁇ precursor and the 24 kDa mature dimer are present in the soft protein corona of the non-imprinted and imprinted nanoparticles of both the formulations. However, conversely to what was observed previously for TGF-bB, there is no clear indication of preferential binding to TGF-bI by the imprinted formulations.
  • VEGF Vascular endothelial growth factor
  • Figure 5 the presence of Vascular endothelial growth factor (VEGF) among the protein corona of the nanoparticles ( Figure 5) was determined. The results did not show any preferential affinity for the imprinted or non-imprinted formulations.
  • the putative bands corresponding to the isoforms of VEGF-121, VEGF-165, and VEGF-189, at 17 kDa, 24 kDa, and 48 kDa respectively are distinguishable among the soft protein corona of the NINP1 and MINP1 formulations. In NINP6 and MINP6 formulations, only the VEGF-165 and VEGF-189 isoforms bands were visible.
  • Figure 5 shows the result of the Western blot analysis to determine the presence of VEGF among the protein corona of NINP1/MINP1 and NINP6/MINP6 after incubation in a 25% PL solution. Lanes from left to right: molecular weight ladder; soft protein corona of (C); hard protein corona of after 3, 6 and 10 washing steps.
  • quantification of TGF-bB adsorption by MINP/NINP6 formulation was determined by Dot Blot analysis.
  • NINP/MINP6 formulation was incubated with recombinant human TGF-bB. Several washing cycles was performed to remove loosely adsorbed material. Dot Blot was then used to quantify the mass of GF that specifically bound to these nanoparticles. ImageJ was used to quantify the immunostaining signal intensity, and this was converted to protein concentration based on a standard curve. It was possible to observe that there was a basal level of unspecific adsorption for NINP6 of around 5 ng per mg of NINP. This persisted regardless of the number of washing cycles.
  • Figure 6 shows the results of Dot blot analysis of rhTGF- 3 in the protein corona of NINP6/MINP6.
  • Figure 6a is a representative picture of the observed blot.
  • Figure 6b shows the mass of remaining adsorbed GF after either 3, 6, or 10 washes, per mg of particles (** p ⁇ 0.01, statistical analysis by two-way ANOVA followed by Sidak multiple comparison).
  • Biochemical stimulation by culture media supplementation with bioactive molecules, such as growth factors is a commonly employed strategy in tissue engineering approaches, aiming at guiding phenotypical changes of a cell population of interest (e.g. stem cell differentiation towards a specific lineage).
  • bioactive molecules such as growth factors
  • rh recombinant human
  • human adipose derived stem cells were culture on surfaces coated with MINPs against TGF- 3 under standard culture conditions. It was envisioned that MINPs could effectively capture TGF-bB endogenously secreted by cultured cells. By further sequestering and enhancing the stability of the factor, this strategy would amplify and prolong its biological activity, regulating the autocrine and paracrine cell signalling pathway. Considering the relevance of TGF-b signalling in tendon and cartilage development, as well as common application of TGF- bB in chondrogenic and tenogenic differentiation protocols, cell response was evaluated by gene expression analysis of a panel of markers associated with tendon and cartilage phenotypes.
  • the panel was composed by genes encoding transcription factors (scleraxis, Sox-9) and structural components of the tissues of interest.
  • the data collected is compared with previously obtained results on in vitro differentiation potential of human adipose-derived stem cells under supplementation with rhTGF-bB.
  • the analysis of transcript levels of tendon and cartilage markers was further complemented by protein expression analysis by immunocytochemistry.
  • atomic force microscopy AFM images of functionalized tissue culture coverslips with polydopamine (PDA) and MINP6/NINP6 are presented in Figure 7.
  • Figure 8 shows fluorescence microscopy images of the same surfaces after treatment with rhTGF- 3, and immunostained with a primary antibody specific for this growth factor followed by a fluorescently conjugated secondary antibody. The results show that both surfaces could be homogeneously covered by both types of nanoparticles (NPs).
  • NPs nanoparticles
  • immunostaining shows that only the MINP6-functionalized surfaces were able to recognize and capture rhTGF- 3 on their surface. This shows that the binding is specifically conferred by the imprinting process and not the result of unspecific adsorption.
  • Figure 7 shows AFM images of tissue culture coverslips coated with polydopamine (PDA) and the subsequent MINP6 and NINP6 functionalized surfaces.
  • Figure 8 shows fluorescence microscopy images of tissue culture coverslips functionalized with either MINP6 or NINP6 after treatment with rhTGF-bB, followed by immunolabelling for this protein.
  • humane adipose-derived stem cells were seeded on to culture surfaces coated with MINP6 or NINP6. After 5 days and 10 days of culture, cell lysates were collected for further RNA isolation and gene expression analysis via quantitative Polymerase Chain Reaction (qPCR) ( Figure 9). After 5 days and 8 days of culture, immunocytochemistry was used to analyse specific protein content and localization ( Figures 10 and 11).
  • qPCR was used to analyse a panel of 4 positive markers (genes whose expression is activated by TGF-bB signalling) and 4 negative markers (bone and adipocyte-related genes, not favored by this signalling pathway).
  • positive markers genes whose expression is activated by TGF-bB signalling
  • negative markers bone and adipocyte-related genes, not favored by this signalling pathway.
  • results show a pronounced increase in expression of SOX9 at day 10 in MINP6 surfaces as compared to NIP6.
  • negative markers FABP4 expression remained in constant downregulation between days 5 and 10 in MINP6 surfaces, while increasing significantly at day 10 on NINP6 surfaces.
  • Figure 9 shows the result of qPCR quantification of expression of the genes of interest. Highlighted are genes with significant differences in expression between cells cultured on MINPs and NINPs.
  • MINP6-surfaces were able to prevent some degree of adipogenesis and promote the commitment of hASCs towards the chondrogenic lineage by virtue of their ability to capture TGF-bB secreted by cell thus modulating their autocrine and paracrine signalling.
  • Figure 10 shows fluorescence images of immunocytochemistry assay of hASCs seeded on MINP6- or NINP6-functionalised surfaces. Blue represents nuclei stained with DAPI; red represents the cytoskeleton stained with phalloidin; green represents proteins of interest marked by immunostaining (either SOX9 or SCX).
  • Figure 11 shows the immunostaining fluorescent images of quantification of SCX and SOX9 protein expression levels.
  • synthesis of MINP/NINP was performed.
  • NPs was performed by inverse microemulsion polymerization. Different combinations of monomers and cross-linker (acrylamide, AAm; methacrylic acid, MAc; 2-aminoethyl methacrylate, 2AmEM; and N,N-methylene-bis-acrylamide, BIS) have been used to produce a series of new MINP and NINP. In brief, combinations of monomers and crosslinker were dissolved in ultrapure water and then T6Rb-3 epitope was added to the mixture and agitated for 30 min at room temperature.
  • monomers and crosslinker acrylamide, AAm; methacrylic acid, MAc; 2-aminoethyl methacrylate, 2AmEM; and N,N-methylene-bis-acrylamide, BIS
  • Anionic (AOT) and nonionic (Brij- 30) surfactants were dissolved in deoxygenated hexane, and the aqueous solution of monomers and suspended epitope was dropped into the hexane solution and stirred under a nitrogen atmosphere for 1 h.
  • the polymerization was initiated by adding ammonium persulfate (APS) and Tetramethylethylenediamine (TEMED), and the solution was mixed for 2 h under vacuum protection. After the completion of polymerization, the NPs were precipitated using ethanol. In order to remove the peptide template, surfactants and unreacted monomers, the NPs were washed with ethanol and then centrifuged.
  • NINPs were synthesized by following the same steps, except that the peptide was not added. Table 1 summarizes the MINP/NINP formulations assessed in the present disclosure.
  • Table 1 Synthesis conditions for production of MINP/NINP formulations.
  • the TGF-bX epitope was immobilized on the surface of gold sensors to assess the affinity of the MINPs by surface plasmon resonance (SPR), as described elsewhere 2 , with slight modifications.
  • Gold-coated sensors BioNavis, Finland
  • the gold sensors were inserted into individual glass flasks, covered with 5 mL of 0.2 mg/mL of cysteamine (Sigma-Aldrich, Saint Louis, USA) solution in ethanol and incubated overnight at room temperature with mild shaking for amine functionalization of sensors surface. After thoroughly washing the sensors first with ethanol and then with ultrapure water, the sensors were immersed in 5 mL of 5% glutaraldehyde (solution in PBS) and incubated for 2 h at room temperature. The sensor surface was rinsed again with PBS. Thereafter, a thin layer of 2.5 mg/mL of TGF-bB epitope solution in PBS (200 pL/ sensor) was applied to the sensor surface.
  • cysteamine Sigma-Aldrich, Saint Louis, USA
  • the senor was rinsed with ultrapure water and 200 pL of 0.1 M ethanolamine (Sigma-Aldrich, Saint Louis, USA) in water were applied to the sensor surface to inactivate the unreacted aldehyde groups. The sensors were then rinsed with ultrapure water, dried under a ISh stream and stored at 4 °C until further use.
  • 0.1 M ethanolamine Sigma-Aldrich, Saint Louis, USA
  • MINP/NINP adsorption to TGF-bB epitope was followed in real time with a multiparametric SPR Navi 200 (BioNavis, Finland).
  • the sensor was designed in a Kretschmann configuration where monochromatic light is reflected from the sensing surface over a range of incident angles thereby causing an angle-dependent reflectance minimum that is detected by the photodiode array. All measurements were performed with the 630 nm laser using full angular scans.
  • the gold sensors derivatized with the TGF-bB epitope as described above, were docked in the SPR equipment and the running buffer (lx PBS) was perfused at 30 pL/min, until a stable baseline was reached.
  • NINP and MINP from different formulations were suspended in filtered 25% (v/v) platelet lysate (PL) in phosphate-buffered saline (PBS 7.4) at a concentration of 1 mg/mL
  • PBS 7.4 phosphate-buffered saline
  • the samples were centrifuged for 15 min at 12000 xg. The supernatant was discarded and the pellet resuspended in 1 mL of PBS 7.4.
  • the suspension was then centrifuged for 15 min at 12000 xg to wash the proteins loosely adsorbed on to the MINP/NINP surface. These washing steps were repeated for a total of three, six and ten times.
  • the pellet was resuspended in mili-Q water and loading buffer (8% sodium dodecyl sulfate, 250 mM Tris hydrochoride (pH 6.8), 20% mercaptoethanol, 40% glycerol and 0.05% bromophenol blue) at a 3:1 volume ratio. After 5 min of incubation at 95 °C, the suspension was centrifuged for 15 min at 12000 xg. The supernatant - which contains the desorbed proteins - was frozen until used.
  • loading buffer 8% sodium dodecyl sulfate, 250 mM Tris hydrochoride (pH 6.8), 20% mercaptoethanol, 40% glycerol and 0.05% bromophenol blue
  • a Western blot analysis was performed.
  • the proteins adsorbed to the MINP/NINP after the sequential washes were separated by electrophoresis in a 12.5% separation gel and 4% stacking gel (SDS Gel kit, Sigma-Aldrich, Portugal) according to standard procedures (20 pL of sample were loaded).
  • a molecular weight marker (PageRuler Plus Prestained Protein Ladder, 10 to 250 kDa, Thermo Scientific, United States) was used as reference.
  • the gel was then transferred onto nitrocellulose membranes (GE Healthcare Life Science, Germany) using Thermo Scientific Pierce Power System (program: 25 V, 1 mA, 1 h).
  • the membranes were blocked at room temperature for 1 hour in 5% BSA in tris-buffered saline with tween 20 (TBST) buffer solution. Then, the membranes were incubated overnight at 4°C in primary antibodies solutions against the target protein: anti-TGF-bI (Abeam, Cambridge; 1:5000 dilution in blocking buffer) and anti-TGF- 3 (abl5537, Abeam, Cambridge; 1:2000 in blocking buffer). Thereafter, the membranes were washed with TBST buffer and incubated with an alkaline phosphatase anti-rabbit IgG secondary antibody (Sigma, USA, 1:2000 dilution in blocking buffer) for 1 hour at room temperature.
  • TBST tris-buffered saline with tween 20
  • the anti-mouse VEGF-A antibody (abl71828, Abeam, Cambridge) was diluted in TBST buffer (1:1000) and used to target VEGF.
  • the alkaline phosphatase horse anti-mouse IgG antibody (Baptista Marques, USA; 1:500 dilution in blocking buffer solution) was used as secondary antibody.
  • the target bands were visualized by application of a colorimetric alkaline phosphatase conjugate substrate kit (Bio-Rad, USA) which was applied to the blot, according to the manufacturer's recommendation.
  • NINP6 and MINP6 were suspended in PBS 7.4 at a concentration of 1 mg/ml.
  • rhTGF- 3 was added at a concentration of 0.15 pg/ml. Samples were incubated for 1 h, with mild shaking, at 37 °C. The remaining procedure was the same as the one used to prepare samples for Western Blot.
  • rhTGF- 3 standards were prepared in ultrapure water at concentrations of 21.4 ng/ml, 10.7 ng/ml, 5.35 ng/ml, 2.67 ng/ml, 1.34 ng/ml, and 0.67 ng/ml. 2 pi of each sample and standard were pipetted in triplicates onto the nitrocellulose membranes. The nitrocellulose membranes were dried overnight. Subsequently, the membranes were blocked for 1 h in 5% BSA in TBST solution at room temperature. The membranes were then incubated overnight at 4 °C in primary antibody solution (anti-TGF- 3, abl5537, Abeam, Cambridge; 1:2000 in blocking buffer). Next, the membranes were washed with TBST solution and incubated with an alkaline phosphatase-conjugated anti-rabbit secondary antibody (Sigma, USA, 1:2000 dilution in TBST) at room temperature for 1 hour.
  • Results were visualized by application of a colorimetric alkaline phosphatase conjugate substrate kit (Bio-Rad, USA) which was applied to the blot, according to the manufacturer's recommendation. Signal intensity was quantified using ImageJ software. A calibration curve to relate intensity and protein mass was fit to the standards and used to calculate the TGF- 3 mass in each sample.
  • MIPs epitope-imprinted
  • NIPs non- imprinted surfaces
  • culture surfaces prior to cell seeding, culture surfaces were immersed for 2 hours in complete culture medium, composed by Minimum Essential Medium (a- MEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco) and 1% (v/v) antibiotic/antimycotic solution (Gibco).
  • a- MEM Minimum Essential Medium
  • FBS fetal bovine serum
  • Gibco antibiotic/antimycotic solution
  • human liposuction aspirates were obtained from healthy female donors under informed consent and according to protocols approved by the Ethical Committee of Hospital da Prelada (Porto, Portugal).
  • Human adipose-derived stem cells (hASCs) were isolated from lipoaspirates according to a standard protocol with collagenase (Sigma Aldrich) digestion. Cells were plated in complete culture medium and incubated in a humidified atmosphere at 37 °C with 5% CO2. Culture medium was replaced every 3 days and cells were sub-cultured at 80% confluence.
  • hASCs in passage 3 were seeded at a density of 2.5 x 103 cells/ cm2 on culture surfaces coated with MI NPs or NINPs and preincubated with culture medium, as described previously. Cell attachment and morphology was monitored by light microscopy. After 5 days and 10 days of culture, cells were fixed with 10% neutral buffered formalin (Thermo Fisher Scientific) and cell lysates were collected using TRI Reagent ® (Sigma Aldrich) for subsequent protein and gene expression analysis.
  • RNA extraction, reverse transcription and qPCR were performed.
  • qPCR real-time quantitative polymerase chain reaction
  • Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Tyrosine 3-Monooxygenase/Tryptophan 5- Monooxygenase Activation Protein Zeta (YWHAZ) were chosen as reference genes due to stability of their expression across the sample sets. All values were first normalized to average transcript levels of GAPDH and YWHAZ, and then normalized to samples collected at the moment of cell seeding. Results are displayed as fold changes and each value represents the mean ⁇ standard deviation (SD) of three independent samples obtained under the same conditions.
  • SD standard deviation
  • 3D cell cultures with MIP promote the deposition of chondrogenic-related matrix components was performed.
  • pellets cultured with MIP6 grew to larger sizes than those with NINP6, with an increase of over 43 % in terms of section area ( Figure 12 c).
  • histological slides were stained with Masson trichrome ( Figure 12 b, MT). It is possible to observe that pellets cultured with MINP6 produced significantly more collagenous matrix (stained blue), occupying over 50 % of the section area, contrasting with only around 30 % for NINP6 samples ( Figure 12 d). Alcian blue was used to detect GAGs deposited in the new matrix ( Figure 12 b, AB).
  • pellets cultured with MINP6 produced significantly more GAGs than those cultured with NINP6, as would be expected following TGF-bB signaling. Nevertheless, when normalized by total section area, no significant differences are noted.
  • immunohistochemistry was employed to visualize collagen II (Col II) and aggrecan (Agg). While no differences were observed for Agg, deposition of Col II was markedly higher in MINP6-cultured pellets, which corroborates the results of trichrome staining ( Figure 12 b, Col II).
  • histological results substantiate previous outcomes from 2D cultures. Together with immunocytochemistry and gene expression analysis, results demonstrate that the binding affinity of MINP6 for TGF-bB has the potential to direct hASC fate commitment, shifting their gene expression and protein synthesis in a controlled manner.

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Abstract

La présente invention concerne l'utilisation de nanoparticules ou microparticules de polymère à empreinte moléculaire (MIP) en tant que ligands sélectifs pour des biomacromolécules, à savoir des protéines bioactives combinées à un substrat approprié pour la culture de cellules souches. Les substrats décorés par MIP se lient sélectivement et séquestrent les protéines bioactives imprimées d'origine exogène ou endogène, amplifiant et induisant des réponses de cellules souches spécifiques, telles que la survie, la prolifération et la différenciation. La présente invention est utile dans l'ingénierie tissulaire et la médecine régénérative.
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Citations (3)

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WO2001030856A1 (fr) 1999-10-23 2001-05-03 Cranfield University Polymeres a empreinte moleculaire obtenus par polymerisation de matrice
WO2014113573A1 (fr) 2013-01-17 2014-07-24 The Regents Of The University Of California Hydrogels à séquestration et présentation de facteur de croissance
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WO2001030856A1 (fr) 1999-10-23 2001-05-03 Cranfield University Polymeres a empreinte moleculaire obtenus par polymerisation de matrice
WO2014113573A1 (fr) 2013-01-17 2014-07-24 The Regents Of The University Of California Hydrogels à séquestration et présentation de facteur de croissance
CN108785743A (zh) * 2018-06-15 2018-11-13 天津工业大学 一种可诱导干细胞软骨分化的双模板分子印迹高强度水凝胶的制备方法

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