Photocurable Methacrylate Derivatives of Polylactide: a Two-Stage Synthesis in Supercritical Carbon Dioxide and 3D Laser Structuring

Article Photocurable Methacrylate Derivatives of Polylactide: A Two-Stage Synthesis in Supercritical Carbon Dioxide and 3D Laser Structuring

Vladislav S. Kaplin 1,* , Nikolay N. Glagolev 1, Valentina T. Shashkova 1, Irina A. Matveeva 1, Ilya V. Shershnev 1, Tatyana S. Zarkhina 1, Nikita V. Minaev 2 , Nadezhda A. Aksenova 1,3, Boris S. Shavkuta 2,3, Evgeny A. Bezrukov 4, Aleksandr S. Kopylov 1,5, Daria S. Kuznetsova 6, Anastasiia I. Shpichka 3,7 Peter S. Timashev 1,3,7 and Anna B. Solovieva 1 1 Semenov Federal Research Center of Chemical Physics, 4 Kosygin St., 119991 Moscow, Russia; [email protected] (N.N.G.); [email protected] (V.T.S.); [email protected] (I.A.M.); [email protected] (T.S.Z.); [email protected] (N.A.A.); [email protected] (A.S.K.); [email protected] (I.V.S.); [email protected] (P.S.T.); [email protected] (A.B.S.); 2 Research center “Crystallography and Photonics”, Institute of Photonic Technologies, 2 Pionerskaya St., Troitsk, 108840 Moscow, Russia; [email protected] (N.V.M.); [email protected] (B.S.S.) 3 Institute for Regenerative Medicine, Sechenov University, 8 Trubetskaya St., 119991 Moscow, Russia; [email protected] 4 Institute for Urology and Reproductive Health, Sechenov University, 8 Trubetskaya St., 119991 Moscow, Russia; [email protected] 5 Institute of Fine Chemical Technologies, Russian Technological University, 78 Vernandsky Avenue, 119454 Moscow, Russia 6 Institute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research Medical University, Minin and Pozharsky Sq. 10/1, 603950 Nizhny Novgorod, Russia; [email protected] 7 Chemistry Department, Lomonosov Moscow State University, 1-3 Leninskiye Gory, 119991 Moscow, Russia * Correspondence: [email protected]

Received: 17 September 2020; Accepted: 23 October 2020; Published: 29 October 2020

Abstract: A two-stage polylactide modification was performed in the supercritical carbon dioxide medium using the urethane formation reaction. The modification resulted in the synthesis of polymerizable methacrylate derivatives of polylactide for application in the spatial 3D structuring by laser stereolithography. The use of the supercritical carbon dioxide medium allowed us to obtain for the first time polymerizable oligomer-polymer systems in the form of dry powders convenient for further application in the preparation of polymer compositions for photocuring. The photocuring of the modified polymers was performed by laser stereolithography and two-photon crosslinking. Using nanoindentation, we found that Young’s modulus of the cured compositions corresponded to the standard characteristics of implants applied in regenerative medicine. As shown by thermogravimetric analysis, the degree of crosslinking and, hence, the local stiffness of scaffolds were determined by the amount of the crosslinking agent and the photocuring regime. No cytotoxicity was observed for the structures.

Keywords: polylactide; supercritical carbon dioxide; photopolymerization; two-photon polymerization; tissue engineering

1. Introduction Polymeric biocompatible materials have been in special demand in the new ﬁeld of medical materials science, tissue engineering, associated with reconstructive surgery and the development of artiﬁcial organs [1]. Indeed, to design a new generation of implants applied in the replacement of lost

Polymers 2020, 12, 2525; doi:10.3390/polym12112525 www.mdpi.com/journal/polymers Polymers 2020, 12, x FOR PEER REVIEW 2 of 21

Polymeric biocompatible materials have been in special demand in the new field of medical Polymers 2020, 12, 2525 2 of 19 materials science, tissue engineering, associated with reconstructive surgery and the development of artificial organs [1]. Indeed, to design a new generation of implants applied in the replacement of orlost damaged or damaged tissues tissues and organs, and oneorgans, needs one materials needs with materials variable with physicomechanical variable physicomechanical characteristics, whichcharacteristics, cause a minimumwhich cause tissue a reactionminimum [2– 4tissue]. One reaction type of such[2–4]. material One type is polyesters of such ofmaterial aliphatic is hydrocarboxylicpolyesters of aliphatic acids, firsthydrocarboxylic of all, polylactide acids, acid first (polylactide, of all, polylactide PLA) (Figure acid (polylactide,1). Due to the PLA) specifics (Figure of its1). productionDue to the specifics and destruction, of its production PLA occupies and destru one ofction, the PLA basic occupies places in one the of series the basic of biocompatible places in the andseries biodegradable of biocompa polymerstible and usedbiodegrada in regenerativeble polymers medicine used and in plasticregene surgeryrative medicine for reconstruction and plastic of bonesurgery and for cartilage reconstruction defects [ 5of,6 ].bone Besides, and PLAcartilage is a thermoplastic defects [5,6]. polymerBesides, andPLA may is bea thermoplastic processed by extrusion,polymer and molding, may be and processed blow molding. by extrusion, However, molding, when using and PLAblow as molding. an initial However, polymer matrix when forusing the implantPLA as an design, initial it polymer becomes matrix necessary for the to alterimplant a number design, of it itsbecomes characteristics, necessary first to alter of all, a itsnumber fragility, of lowits characteristics, porosity, low thermalfirst of stability, all, its andfragility, high hydrophobicity,low porosity, leadinglow thermal to low cellstability, adhesion and tohigh the material’shydrophobicity, surface leading [7,8]. to low cell adhesion to the material’s surface [7,8].

Figure 1. The structural formula of poly(lactic) acid (PLA).

To improveimprove cell cell adhesion adhesion and increaseand increase hydrophilicity hydrophilicity and bactericidal and bactericidal properties ofproperties polylactides, of apolylactides, laser or plasma a laser treatment or plasma of treatment the polymer of the materials’ polymer surfacematerials’ is usedsurface [9 ,is10 ],used with [9,10], the additionwith the ofaddition crosslinking of crosslinking agents carryingagents carrying reactive reacti groups—carboxyl,ve groups—carboxyl, hydroxyl, hydr aminooxyl, amino groups, groups, or their or combinationtheir combination [11–13 [11–13],], as well as aswell grafting as grafting of polymers of polymers with antibacterialwith antibact propertieserial properties [14]. [14]. The bulk properties of PLA, in particular, hydrophobicity,hydrophobicity, may be variedvaried byby synthesizingsynthesizing copolymers of polylactide with carbonates, lactones, glycolides, glycols, urethanes, etc. [15], [15], or by graft-copolymerization, e.g., introduction of monomeric lactide in a hydrophilichydrophilic polyethyleneglycolpolyethyleneglycol macromolecule [[16].16]. Chemical modificationmodification of of the the PLA PLA terminal terminal groups groups may appear may appear a promising a promising approach toapproach enhancing to PLAenhancing hydrophilicity PLA hydrophilicity and affinity and to affinity cells with to cells the simultaneouswith the simultaneous decrease ofdecrease the polymer of the polymer fragility. Copolymersfragility. Copolymers of lactic acidof lactic with acid hydrophilic with hydrophilic monomers monomers are usually are synthesized usually synthesized for this purpose.for this Inpurpose. particular, In particular, PLA copolymers PLA copolymers with starch with are starch obtained are in obtained toluene in at 150toluene◦C, followedat 150 °С by, followed azeotropic by dehydrationazeotropic dehydration [17], or by extrusion[17], or atby 180 extrusion◦C[18]. PLAat 180 copolymers °С [18]. with PLA polyethyleneoxide copolymers with are synthesizedpolyethyleneoxide in toluene are in synthesized a nitrogen atmosphere in toluene atin 85a ◦nitrogenC[19]. One atmosphere of the most at promising 85 °С [19]. approaches One of the to themost structuring promising of approaches polylactide (asto the well structuring as other polymers of polylactide containing (as hydroxylwell as other groups, polymers e.g., chitosan containing [20]), inhydroxyl which modificationgroups, e.g., of chitosan the PLA [20]), terminal in which groups modification is applied, isof the the utilization PLA terminal of various groups techniques is applied, of thermalis the andutilization photocuring, of various including techniques laser stereolithography of thermal [ 21and]. To accomplishphotocuring, this, including new functional laser groupsstereolithography containing unsaturated[21]. To accomplish bonds are this, added new to PLAfunctional hydroxyl groups groups contai vianing esterification unsaturated [22]. Asbonds the modifyingare added agents,to PLA anhydride hydroxyl [23groups,24] or via chloroanhydride esterification [22]. of methacrylic As the modifying acid [25] areagents, most anhydride frequently used.[23,24] Such or chloroanhydride reactions proceed of in themethacrylic presence ofacid pyridine [25] are [26 ]most or trimethylamine frequently used. [27, 28Such] in thereactions liquid medium:proceed in in the toluene presence [29], of dimethylformamide pyridine [26] or trimet [26],hylamine or haloalkanes [27,28] [30 in]. the One liquid may alsomedium: graft thein toluene double bonds[29], dimethylformamide to polyesters of hydroxyacids [26], or usinghaloalkanes a more advanced[30]. One approachmay also of urethanegraft the formation,double bonds with theto usepolyesters of diisocyanates of hydroxyacids and hydroxyl-containing using a more advanced modifying approach agents of [ 31urethane,32]. However, formation, this with method the isuse only of scantilydiisocyanates described and inhydroxyl-containing the literature, though modifying such reactions agents do[31,32]. not require However, toxic this substances, method suchis only as anhydridesscantily described and chloroanhydrides in the literature, ofthough acids, such tertiary reactions amines, do or not pyridine require [ 33toxic]. This substances, fact is probably such as relatedanhydrides to the and diffi chloroanhydridesculties arising due of to acids, a higher tertiary sensitivity amines, of or urethane pyridine formation [33]. This to fact the presenceis probably of evenrelated insignificant to the difficulties moisture, arising with due the to need a higher to dry se thensitivity reactants of urethane and the formation solvent and to the to conductpresence the of reactioneven insignificant in an inert moisture, atmosphere. with the need to dry the reactants and the solvent and to conduct the reactionEarlier, in an we inert performed atmosphere. a modification of polylactide with the introduction of polymerizable acrylate groupsEarlier, into the we macromolecule performed a viamodification the reactions of ofpolylactide esterification with and the urethane introduction formation of polymerizable of the terminal carboxylacrylate groups and hydroxyl into the groups macromol in a tolueneecule via or the methylene reactions chloride of esterifi solution,cation respectively,and urethane which formation allowed of obtaining 3D crosslinked structures by photopolymerization [29,34]. It should also be noted that all Polymers 2020, 12, 2525 3 of 19 the above-mentioned ways of synthesis in the liquid phase involve the stage of product separation: reprecipitation, centrifugation, washing, and long drying. This leads not only to product losses but also to obtaining a resin-like mass as a result, inconvenient for further application since it is almost impossible to remove the solvent completely in common conditions. As reported in [22,35], a partial self-crosslinking takes place in such a case, resulting in the shelf life of the product not exceeding 1–2 months. We showed earlier that the esterification reaction in a solution had a degree of conversion limited by 65% [29]. When using urethane formation for PLA modification in a solution, we managed to raise the degree of conversion to 92%; however, the product state as a viscous mass did not allow its laser structuring application due to proceeding self-crosslinking [34]. Carbon dioxide is most frequently used as a supercritical state solvent for conducting various chemical reactions, that is, primarily related to its convenient critical parameters (31.2 ◦C, 7.3 MPa) and affinity to both polar and nonpolar polymer matrices. Besides, supercritical carbon dioxide (scCO2) is capable of dissolving organic molecules of different polarities and disappearing from the system after the process completion that facilitates the separation and purification of products [36,37]. Earlier, using the scCO2 medium, the bulk properties of ultra-high-molecular-weight polyethylene (UHMWPE) were modified by the synthesis of crosslinked methacrylate systems inside the UHMWPE matrix, which formed interpenetrating network structures with interesting mechanical properties [38,39]. In particular, it was noted that mixing UHMWPE with polymethylmethacrylate-co-poly(ethyleneglycol)dimethacrylate resulted in the formation of an extremely strong crosslinked material, which did not exhibit the usual thermal deformation behavior observed for UHMWPE. In the field of creation of biodegradable implants, supercritical CO2 has already found application as a pore-forming agent [40,41] or for sterilization [42]. However, the use of scCO2 as a medium for chemical modification of polylactide and its derivatives has almost no mentions, despite its advantages of inertness and the absence of moisture, rapid removal from the reaction mixture, and absence of toxicity. In this study, supercritical CO2 was used as a solvent for urethane formation for the first time. The study objective was an elaboration of an approach for the synthesis of methacrylate PLA derivatives via the urethane formation reaction (from the interaction of polylactide hydroxyl groups with diisocyanates) in the medium of supercritical carbon dioxide. The reaction was conducted in two stages with the separation of the intermediate diisocyanate PLA derivative, followed by its interaction with ethyleneglycol monomethacrylate, leading to the formation of PLA containing polymerizable methacrylic groups. Using laser stereolithography and two-photon crosslinking, we prepared 3D crosslinked structures based on the synthesized PLA methacrylate and tested them for cytotoxicity.

2. Materials and Methods

2.1. PLA Modification Process PLA methacrylation via the urethane formation reaction was performed with the use of 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate (ITI, 98%, a mixture of isomers, Aldrich, St. Louis, MO, USA) (Figure2A) and ethyleneglycol monomethacrylate (EGM, Aldrich, St. Louis, MO, USA) (Figure2B). Polylactide with the average molecular weight of 5 103 Da (Aldrich, St. Louis, MO, × USA) was used for the modification. All the components were used without additional purification. Dry carbon dioxide, with the volume content of water vapor not exceeding 0.001%, according to the quality certificate, was utilized. The solubility of the initial reactants in the scCO2 medium was tested as follows: each of the reactants was placed into a steel reactor with a volume of 3 mL and quartz windows, in a concentration corresponding to its concentration in the reaction. In the reactor, we reproduced the scCO2 medium at the temperature and pressure corresponding to the reaction conditions. Then, we registered a UV-Vis absorption spectrum using a Cary 50 spectrophotometer Polymers 2020, 12, 2525 4 of 19

(Agilent Technologies, Santa Clara, CA, USA) and compared the acquired spectrum with the absorption Polymers 2020, 12, x FOR PEER REVIEW 4 of 21 spectrum ofPolymers the 2020 same, 12, x substance FOR PEER REVIEW in chloroform. 4 of 21 As mentionedthe reaction above, conditions. the synthesisThen, we ofregistered the methacrylate a UV-Vis absorption PLA derivative spectrum in using scCO a2 wasCary conducted50 in the reaction conditions. Then, we registered a UV-Vis absorption spectrum using a Cary 50 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) and compared the acquired two stages.spectrophotometer At the first stage, (Agilent PLA isocyanateTechnologies, (Figure Santa 3Clara,) was CA, obtained USA) byand the compared interaction the acquired of PLA hydroxyl spectrum with the absorption spectrum of the same substance in chloroform. groups withspectrum ITI isocyanate with the absorption groups spectrum as follows: of the 0.001same substance mol of PLAin chloroform. and 0.0011, 0.002, and 0.003 mol of As mentioned above, the synthesis of the methacrylate PLA derivative in scСО2 was conducted ITI (with the excessAs mentioned of 10%, above, 100%, the synthesis and 200%, of the respectively, methacrylate PLA with derivative respect in toscСО PLA),2 was asconducted well as 0.15 mL in two stages. At the first stage, PLA isocyanate (Figure 3) was obtained by the interaction of PLA in two stages. At the first stage, PLA isocyanate (Figure 3) was obtained by the interaction of PLA of the catalyst—dibutyltinhydroxyl groups with dilaurate ITI isocyanate (Aldrich, groups St.as follows: Louis, 0.001 MO, mol USA)—was of PLA and placed 0.0011, 0.002, into aand steel reactor hydroxyl groups with ITI isocyanate groups as follows: 0.001 mol of PLA and 0.0011, 0.002, and 0.003 mol of ITI (with3 the excess of 10%, 100%, and 200%, respectively, with respect to PLA), as well with the volume0.003 mol ofof ITI 78 (with cm thecontaining excess of 10%, a magnetic100%, and 200%, anchor. respectively, After with that, respect thereactor to PLA), wasas well filled with as 0.15 mL of the catalyst—dibutyltin dilaurate (Aldrich, St. Louis, MO, USA)—was placed into a gaseous COas 20.15at mL a pressure of the catalyst—dibutyltin of about 6 MPa dilaurate at 25 (Aldrich,◦C and St. heated Louis, toMO, 40 USA)—was◦C; in doing placed so, into the a pressure steel reactor with the volume of 78 cm3 containing a magnetic anchor. After that, the reactor was increasedsteel to 9 reactor MPa, with and the carbon volume dioxide of 78 cm underwent3 containing a amagnetic transition anchor. first After to that, the liquidthe reactor and was then to the filled with gaseous СО2 at a pressure of about 6 MPa at 25 °С and heated to 40 °С; in doing so, the filled with gaseous СО2 at a pressure of about 6 MPa at 25 °С and heated to 40 °С; in doing so, the supercriticalpressure state increased (the whole to 9 processMPa, and takescarbon about dioxide 8 un min).derwent The a reactiontransition wasfirst to conducted the liquid and for then either 10 or 20 pressure increased to 9 MPa, and carbon dioxide underwent a transition first to the liquid and then to the supercritical state (the whole process takes about 8 min). The reaction was conducted for h, and thento COthe 2supercriticalwas removed. state (the The whole completion process takes of the about first 8 stage min). wasThe reaction controlled was usingconducted IR spectroscopyfor either 10 or 20 h, and then СО2 was removed. The completion of the first stage was controlled using and size-exclusioneither 10 or chromatography20 h, and then СО2 was (SEC) removed. via The the completion appearance of the and first growthstage was ofcontrolled the isocyanate using band IR spectroscopy and size-exclusion chromatography (SEC) via the appearance and growth of the (2268 cm 1IR) untilspectroscopy its intensity and size-exclusion became constant chromatography (Figure (SEC)4) in via the the IR appearance spectrum and of growth the reaction of the mixture −isocyanate band (2268 cm−1) until its intensity became constant (Figure 4) in the IR spectrum of the isocyanate band (2268 cm−1) until its intensity became constant (Figure 4) in the IR spectrum of the purified byreaction reprecipitation, mixture purified which, by reprecipitation, according to which, the SECaccording data to (Figure the SEC5 ),data was (Figure free 5), from was the free unreacted reaction mixture purified by reprecipitation, which, according to the SEC data (Figure 5), was free from the unreacted three-fold excess of ITI (which is usually present at 25.7 min at a chromatogram three-foldfrom excess the unreacted of ITI (which three-fold is usuallyexcess of ITI present (which atis usually 25.7 min present at aat chromatogram 25.7 min at a chromatogram of the unpurified of the unpurified isocyanate polylactide derivative (Figure S1)). isocyanateof polylactide the unpurified derivative isocyanate poly (Figurelactide S1)). derivative (Figure S1)).

Figure 2. Structural formulas of the reactants used: Figure 2. StructuralFigure formulas2. Structural of the reactants formulas used: (A)—3-isocyanatomethyl-3,5,5-trimethylcyclohexylof the reactants used: (A)—3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate (ITI), (B)—ethyleneglycol (A)—3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate (ITI), (B)—ethyleneglycol isocyanatemonomethacrylate (ITI), (B)—ethyleneglycol (EGM). monomethacrylate (EGM). monomethacrylate (EGM).

Polymers 2020, 12Figure, x FOR 3. ThePEER structural REVIEW formula of the isocyanate PLA derivative, obtained in the first stage. 5 of 21 Figure 3. FigureThe structural 3. The structural formula formula of theof the isocyanate isocyanate PL PLAA derivative, derivative, obtained obtained in the first in thestage. ﬁrst stage.

FigureFigure 4. IR 4. IR spectrum spectrum of of the the reactionreaction mixture mixture obtained obtained at the at thefirst ﬁrststage, stage, after its after purification its puriﬁcation by by reprecipitation.reprecipitation.

Figure 5. SEC (size-exclusion chromatography) elution profile of the reaction mixture obtained at the first stage, after its purification by reprecipitation. The red line and triangles indicate the signal boundaries of the main reaction product.

In the second stage, the methacrylate PLA derivative (Figure 6) was obtained by the interaction of isocyanate groups of PLA derivative obtained at the first stage with the EGM hydroxyl groups. EGM in the amount of 0.0011, 0.003, or 0.005 mol, corresponding to the excess of 10%, 200%, or 400%, respectively, to PLA, and 0.15 mL of dibutyltin dilaurate as a catalyst were loaded into the reactor containing the reaction mixture obtained at the first stage (PLA isocyanate and unreacted ITI). The reaction was conducted in the scСО2 medium in the same conditions, like those at the first stage, for 10 h. The completion of the methacrylate PLA derivative formation was also controlled by SEC and IR spectroscopy via the disappearance of the isocyanate band (2268 cm−1) and appearance and growth of the double bond vibration band (1637 cm−1) (Figure 7) in the IR spectrum of the reaction mixture purified by reprecipitation, which, according to the SEC data (Figure 8), was free from the unreacted five-fold excess of EGM (with only one main product present). The reaction mixture after the СО2 removal represented a dry powder, which was dissolved in chloroform and

Polymers 2020, 12, x FOR PEER REVIEW 5 of 21

Figure 4. IR spectrum of the reaction mixture obtained at the first stage, after its purification by Polymers 2020, 12, 2525 5 of 19 reprecipitation.

FigureFigure 5. 5.SEC SEC (size-exclusion (size-exclusion chromatography) chromatography) elution elution profile profile of of the the reaction reaction mixture mixture obtained obtained at at thethe first first stage, stage, after after its its purification purification by by reprecipitation. reprecipitation. The The red red line line and and triangles triangles indicate indicate the the signal signal boundariesboundaries of of the the main main reaction reaction product. product.

InIn the the second second stage, stage, the the methacrylate methacrylate PLA PLA derivative derivative (Figure (Figure6) was 6) was obtained obtained by the by interactionthe interaction of isocyanateof isocyanate groups groups of PLA of derivativePLA derivative obtained obtained at the firstat the stage first with stage the with EGM the hydroxyl EGM hydroxyl groups. EGM groups. in theEGM amount in the of 0.0011,amount 0.003, of 0.0011, or 0.005 0.003, mol, correspondingor 0.005 mol, tocorresponding the excess of 10%, to the 200%, excess or 400%, of 10%, respectively, 200%, or to400%, PLA, respectively, and 0.15 mL to of PLA, dibutyltin and 0.15 dilaurate mL of asdibutyltin a catalyst dilaurate were loaded as a catalyst into the were reactor loaded containing into the thereactor reaction containing mixture the obtained reaction at mixture the first obtained stage (PLA at the isocyanate first stage and (PLA unreacted isocyanate ITI). and The unreacted reaction was conducted in the scCO medium in the same conditions, like those at the first stage, for 10 h. ITI). The reaction was conducted2 in the scСО2 medium in the same conditions, like those at the first Thestage, completion for 10 h. The of the completion methacrylate of the PLA methacrylate derivative PLA formation derivative was formation also controlled was also by controlled SEC and IR by 1 spectroscopySEC and IR spectroscopy via the disappearance via the disappea of the isocyanaterance of the band isocyanate (2268 cm band− ) and (2268 appearance cm−1) and and appearance growth 1 ofand the growth double bondof the vibration double bond band (1637vibration cm− band) (Figure (16377) incm the−1) IR(Figure spectrum 7) in of the the IR reaction spectrum mixture of the purifiedreaction by mixture reprecipitation, purified which,by reprecipitation, according to which, the SEC according data (Figure to the8), wasSEC freedata from (Figure the unreacted8), was free five-foldfrom the excess unreacted of EGM five-fold (with onlyexcess one of main EGM product (with only present). one main The reactionproduct mixturepresent). after The the reaction CO2 removal represented a dry powder, which was dissolved in chloroform and precipitated in a ten-fold mixture after the СО2 removal represented a dry powder, which was dissolved in chloroform and hexane excess for purification, separation, and analysis of the final product. The degree of polylactide modification was measured as follows: a charge of the final product containing both modified and unreacted PLA (ma) was dissolved in dichloromethane, then a photoinitiator and a curing agent (oligourethanedimethacrylate, OUDM) were added, and photocuring was conducted using a scattered light of a mercury lamp according to the procedure described in [34]. Then, the prepared crosslinked composition was extracted with tetrahydrofuran of a volume Ve for 24 h. As a result, an extract of a volume Ve was prepared, containing the entire unmodified PLA, which did not participate in the photocuring reaction. Using a calibration plot of the PLA peak area vs. PLA concentration, we calculated the unmodified PLA concentration in the extract (C). Then, the overall weight of the unmodified PLA extracted from the photocured charge was calculated (C*Ve). Based on the obtained values, the degree of PLA modification was calculated according to Formula (1).

(ma m )/ma 100% (1) − b × Polymers 2020, 12, x FOR PEER REVIEW 6 of 21 Polymers 2020, 12, x FOR PEER REVIEW 6 of 21 precipitated in a ten-fold hexane excess for purification, separation, and analysis of the final precipitated in a ten-fold hexane excess for purification, separation, and analysis of the final product. The degree of polylactide modification was measured as follows: a charge of the final product. The degree of polylactide modification was measured as follows: a charge of the final product containing both modified and unreacted PLA (ma) was dissolved in dichloromethane, then product containing both modified and unreacted PLA (ma) was dissolved in dichloromethane, then a photoinitiator and a curing agent (oligourethanedimethacrylate, OUDM) were added, and a photoinitiator and a curing agent (oligourethanedimethacrylate, OUDM) were added, and photocuring was conducted using a scattered light of a mercury lamp according to the procedure photocuring was conducted using a scattered light of a mercury lamp according to the procedure described in [34]. Then, the prepared crosslinked composition was extracted with tetrahydrofuran described in [34]. Then, the prepared crosslinked composition was extracted with tetrahydrofuran of a volume Ve for 24 h. As a result, an extract of a volume Ve was prepared, containing the entire of a volume Ve for 24 h. As a result, an extract of a volume Ve was prepared, containing the entire unmodified PLA, which did not participate in the photocuring reaction. Using a calibration plot of unmodified PLA, which did not participate in the photocuring reaction. Using a calibration plot of the PLA peak area vs. PLA concentration, we calculated the unmodified PLA concentration in the the PLA peak area vs. PLA concentration, we calculated the unmodified PLA concentration in the extract (С). Then, the overall weight of the unmodified PLA extracted from the photocured charge extract (С). Then, the overall weight of the unmodified PLA extracted from the photocured charge was calculated (С*Ve). Based on the obtained values, the degree of PLA modification was calculated was calculated (С*Ve). Based on the obtained values, the degree of PLA modification was calculated according to Formula (1). according to Formula (1). Polymers 2020, 12, 2525 (ma − mb)/ma × 100% 6 of (1) 19 (ma − mb)/ma × 100% (1)

Figure 6. The structural formula of the methacrylate PLA derivative, obtained at the second stage. Figure 6. The structural formula of the methacrylate PLAPLA derivative, obtained at thethe secondsecond stage.stage.

Figure 7. IRIR spectrum spectrum of of the the reaction reaction mixture mixture obtained obtained at atthe the second second stage, stage, after after its itspurification puriﬁcation by PolymersFigure 2020 7., 12 IR, x spectrumFOR PEER ofREVIEW the reaction mixture obtained at the second stage, after its purification by 7 of 21 byreprecipitation. reprecipitation. reprecipitation.

FigureFigure 8. 8.SEC SEC elution elution proﬁle profile of the of reaction the reaction mixture obtainedmixture atobtained the second at stage,the second after its stage, puriﬁcation after byits reprecipitation.purification by Thereprecipitation. red line and The triangles red line indicate and tr theiangles signal indicate boundaries the signal of the boundaries main reaction of product.the main reaction product. The analysis of the products was conducted by SEC with a Waters chromatograph (Milford, MA, USA)The analysis (Breeze of system, the products Ultrasilicagel was conducted 100 Å, 500 byÅ, SEC and with 1000 a ÅWaters standard chromatograph columns, 1 mL(Milford,/min, MA, USA) (Breeze system, Ultrasilicagel 100 Å, 500 Å, and 1000 Å standard columns, 1 mL/min, detectors—a refractometer and a UV-module with a variable wavelength) and FTIR spectroscopy (Varian 800, Agilent Technologies, Santa Clara, CA, USA). To confirm the formation of the isocyanate and methacrylate PLA derivatives, we also conducted an NMR study. 1Н- and 13С-NMR spectra of the half-product and product were acquired with a Bruker Avance 600 spectrometer (Billerica, MA, USA). To purify the substances from the unreacted reactants and side-products, they were dissolved in dichloromethane with the following reprecipitation in a ten-fold excess of hexane. The spectra were registered in a CDCl3 solution at the temperature of 25 °С and the frequency of 600 MHz (1Н-NMR) and 150 MHz (13С-NMR). PLA isocyanate derivative: 1H-NMR (CDCl3, 600 MHz) δ, ppm: 0.88 (t, 9H, CH3c,d), 0.93–1.11 (15H, CH3c,d, CH2e), 1.26–1.30 (m, 6H, CH2e), 1.47–1.66 (m, 3H, CH3b), 2.37 (m, 1H, CHh), 3.05 (s, 2H, CH2f), 4.73 (m, 1H, NHg), 5.13–5.24 (m, 1H, CHa). PLA methacrylate derivative: 1H-NMR (CDCl3, 600 MHz) δ, ppm: 0.88 (t, 9H, CH3c,d), 0.93–1.11 (15H, CH3c,d, CH2e), 1.25–1.31 (m, 6H, CH2e), 1.47–1.60 (m, 3H, CH3b), 1.95 (s, 3H, CH3n), 2.32 (t, 1H, CHh), 2.92 (m, 2H, CH2f), 4.32 (m, 4H, CH2j,k), 4.63 (d, 1H, NHg), 4.86 (t, 1H, NHi), 5.13–5.24 (m, 1H, CHa), 5.59 (s, 1H, CH2m,l), 6.14 (s, 1H, CH2m,l).

2.2. Preparation of Crosslinked Structures The crosslinked samples represented either films with a diameter of 8–10 mm (and the film thickness of 1 mm) or scaffolds. To obtain films, a DRT-1000 mercury lamp (with the intensity of incident unfiltered light I ≈ 0.03 W/cm2, exposure of 300 s) [34] or a powerful (50 W) light emitting diode (LED) matrix (Epileds Technologies Inc., Tainan, Taiwan, λ = 365 nm, I = 5 W/сm2, exposure of 200 s, out-of-focus light) were used. The photoactive composition was a 12% PLA methacrylate solution in methylene chloride containing a crosslinking agent—oligourethanedimethacrylate (OUDM) (2.3–20 wt. %)—and a photoinitiator (Michler’s ketone (Aldrich, St. Louis, MO, USA), PI, 5 wt. % with respect to the weight of the modified PLA). The composition (in the form of a viscous liquid with a volume of about 0.3 mL) was placed onto Teflon support (3 cm × 3 cm). It appeared possible to use the unprecipitated reaction mixture with the addition of the deficient amount of the crosslinker and photoinitiator as a photoactive composition.

Polymers 2020, 12, 2525 7 of 19 detectors—a refractometer and a UV-module with a variable wavelength) and FTIR spectroscopy (Varian 800, Agilent Technologies, Santa Clara, CA, USA). To conﬁrm the formation of the isocyanate and methacrylate PLA derivatives, we also conducted an NMR study. 1H- and 13C-NMR spectra of the half-product and product were acquired with a Bruker Avance 600 spectrometer (Billerica, MA, USA). To purify the substances from the unreacted reactants and side-products, they were dissolved in dichloromethane with the following reprecipitation in a ten-fold excess of hexane. The spectra were registered in a CDCl3 solution at the temperature of 25 ◦C and the frequency of 600 MHz (1H-NMR) and 150 MHz (13C-NMR). 1 PLA isocyanate derivative: H-NMR (CDCl3, 600 MHz) δ, ppm: 0.88 (t, 9H, CH3c,d), 0.93–1.11 (15H, CH3c,d, CH2e), 1.26–1.30 (m, 6H, CH2e), 1.47–1.66 (m, 3H, CH3b), 2.37 (m, 1H, CHh), 3.05 (s, 2H, CH2f), 4.73 (m, 1H, NHg), 5.13–5.24 (m, 1H, CHa). 1 PLA methacrylate derivative: H-NMR (CDCl3, 600 MHz) δ, ppm: 0.88 (t, 9H, CH3c,d), 0.93–1.11 (15H, CH3c,d, CH2e), 1.25–1.31 (m, 6H, CH2e), 1.47–1.60 (m, 3H, CH3b), 1.95 (s, 3H, CH3n), 2.32 (t, 1H, CHh), 2.92 (m, 2H, CH2f), 4.32 (m, 4H, CH2j,k), 4.63 (d, 1H, NHg), 4.86 (t, 1H, NHi), 5.13–5.24 (m, 1H, CHa), 5.59 (s, 1H, CH2m,l), 6.14 (s, 1H, CH2m,l).

2.2. Preparation of Crosslinked Structures The crosslinked samples represented either films with a diameter of 8–10 mm (and the film thickness of 1 mm) or scaffolds. To obtain films, a DRT-1000 mercury lamp (with the intensity of incident unfiltered light I 0.03 W/cm2, exposure of 300 s) [34] or a powerful (50 W) light emitting ≈ diode (LED) matrix (Epileds Technologies Inc., Tainan, Taiwan, λ = 365 nm, I = 5 W/cm2, exposure of 200 s, out-of-focus light) were used. The photoactive composition was a 12% PLA methacrylate solution in methylene chloride containing a crosslinking agent—oligourethanedimethacrylate (OUDM) (2.3–20 wt. %)—and a photoinitiator (Michler’s ketone (Aldrich, St. Louis, MO, USA), PI, 5 wt. % with respect to the weight of the modified PLA). The composition (in the form of a viscous liquid with a volume of about 0.3 mL) was placed onto Teflon support (3 cm 3 cm). It appeared possible to use × the unprecipitated reaction mixture with the addition of the deficient amount of the crosslinker and photoinitiator as a photoactive composition. Scaffolds were prepared by laser stereolithography [43] and two-photon crosslinking [44,45]. Samples with the volume of 60 µL were fabricated by layer-by-layer deposition of 15 µL of the photoactive composition on a coverslip. The air-dried composition contained approximately 10 wt. % of the residual solvent. The crosslinked samples after photocuring were kept in tetrahydrofuran for a day to remove the unreacted polymer, as well as unreacted reactants and side products of the reaction, then dried on air.

2.3. Studies of Mechanical Characteristics Local Young’s moduli of crosslinked samples were measured with a Piuma Nanoindenter (Optics11, Amsterdam, The Netherlands) using a cantilever with a spring constant of 52.4 N/m and a tip radius of 30.0 µm. The measurements were conducted on air. The area of Young’s modulus maps was 300 300 µm2 with an increment of 30 µm by X and Y axes. Based on the measurement results, × the effective Young’s modulus (E) of a sample was calculated (mean standard deviation (SD)). ± 2.4. Differential Thermal Analysis For the differential thermal analysis (DTA), a synchronous STA 449 F3 thermal analyzer (NETZCH, Selb, Deutschland) was used. The destruction process was conducted on air at the gas flow rate of 30 mL/min and a linear heating rate of 10 ◦C/min, and the samples’ charges were 2–5 mg. The weight 3 losses were registered with the accuracy up to 10− mg, and the relative error of temperature measurement was 1.5 C. The weight loss (thermogravimetric analysis, TGA) and the rate of the ± ◦ weight loss (W) dependencies on the temperature were studied. As quantitative characteristics of the proceeding thermooxidative destruction of PLA methacrylate derivatives, obtained by different Polymers 2020, 12, 2525 8 of 19

photocuring techniques, we used the weight loss (∆m, %), maximum destruction rate (WMax,%/min), and coke residue (coke, wt. %).

2.5. Cell Culture For cytotoxicity assays, we used a 3T3 mouse ﬁbroblast cell line (Biobank, Sechenov University, Moscow, Russia) Cells were cultured, as described elsewhere [46,47]: under standard conditions (37 ◦C, 5% CO2) in DMEM/F12 (1:1) basal medium (Gibco) supplemented with a 10% FBS (HyClone) and 1% penicillin-streptomycin (Gibco). Every 2–3 days, we changed the medium. Cells were passaged when they reached 80% conﬂuency.

2.6. Cytotoxicity Assessment The possible cytotoxicity of the synthesized material was assessed using Live/Dead (Sigma Aldrich, St. Louis, MO, USA) staining and AlamarBlue (Invitrogen, Carlsbad, CA, USA) and MTT (Sigma Aldrich, St. Louis, MO, USA) assays. To reveal live and dead cells, we seeded scaﬀolds with the previously described design [48–50] with 1 105 cells (per scaﬀold) and stained them for 72 h × with calcein-AM (live, green) and propidium iodide (dead, red) (Sigma Aldrich, St. Louis, MO, USA); cell nuclei were additionally stained with Hoechst 33342 (blue). For AlamarBlue and MTT assays, we inoculated 5 103 cells to each well in a 96-well plate and added a matrix extract or sodium × dodecyl sulfate (positive control, in seven dilutions) in 24 h. The matrix extract was prepared by dipping its part with the surface area of 6 cm2 into 1 mL DMEM/F12 supplemented with 5% FBS and 1% penicillin-streptomycin and placing for 24 h in a thermostat (ISO 10993-12:2012). The AlamarBlue and MTT assays were performed in accordance with the manufacturer’s instructions.

3. Results and Discussion

3.1. A Two-Stage Synthesis of Polylactide Modified with Functional Groups in the scCO2 Medium It is known that one may introduce unsaturated methacrylic groups to macromolecules containing reactive hydroxyl groups (polyols, glycols, and hydroxycarboxylic acids) via the reaction of urethane formation performed in one or two stages [51]. The single-stage synthesis is the simplest way of modification; however, the lower reactivity of PLA terminal hydroxyl groups with respect to the EGM hydroxyl group leads to a competitive reaction of the interaction between EGM and ITI. Taking into account this circumstance, to increase the degree of PLA modification, we performed methacrylation in the supercritical carbon dioxide medium in two stages, obtaining the isocyanate PLA derivative at the first stage. All the initial reactants, including polylactide, as well as intermediate and final products, are shown to be soluble in the scCO2 medium; thus, the reaction in scCO2 proceeded in the homogeneous conditions. To boost the degree of modification of the methacrylate polylactide derivative, we used a molar excess of ITI (by 1.1, 2.0, and 3.0 times at the first stage) and EGM (by 1.1, 3.0, and 5.0 times at the second process stage) with respect to PLA. The reaction time for the PLA isocyanate derivative synthesis is also increased from 10 to 20 h. It should be noted that usually when obtaining isocyanate derivatives of polymers containing hydroxyl groups, a 10% excess of the isocyanate and methacrylating agent is used [34]. However, as follows from Figure9, at such ratios of components, the degree of modification of methacrylated PLA is only 20% due to the low reactivity of PLA terminal hydroxyl groups. In the case of the increased ITI and, hence, EGM content in the system, the degree of modification of methacrylated PLA grows (Figure9). At the same time, the excess of the methacrylating agent (EGM) with respect to the isocyanate component is related to the necessity of binding toxic isocyanate groups not having reacted with PLA. It should be noted that the increase of the ITI and EGM content in the system above the mentioned ratios does not result in the increase of the methacrylated PLA degree of modification, probably, due to a dramatic viscosity rise. Polymers 2020, 12, 2525 9 of 19

The reaction mixture obtained at the first stage of the process and consisting of PLA isocyanate and unreacted ITI is introduced into the reaction with EGM, which results in the formation of the PLA methacrylate derivative (Figure9). Thus, the use of the large excess of ITI and EGM and the two-stage design of the reaction allows achieving an 82% degree of PLA modification in the scCO2 medium. The obtained degree of conversion is the mean value after the no less than ten-fold experiment reproduction and is as high as those in alternative reactions of PLA methacrylation [26–28]. However, in the scheme, we suggested there is no stage of solvent removal, which is unavoidable when the reaction is conducted in the liquid phase. After the CO2 release, the final reaction mixture represents a stable dry powder, which may be stored for no less than a year without spontaneous self-crosslinking. Secondly, as will be shown further, the reaction mixture does not require additional purification after Polymersthe second 2020,stage 12, x FOR and PEER may REVIEW be used immediately as a base for a photoactive composition. 10 of 21

Figure 9. Average methacrylated PLA degree of modification in single-stage and two-stage reactions of Figure 9. Average methacrylated PLA degree of modification in single-stage and two-stage PLA modification in the scCO2 (supercritical carbon dioxide) medium (40 ◦C, 9 MPa) at different ratios reactions of PLA modification in the scCO2 (supercritical carbon dioxide) medium (40 °С, 9 MPa) at of initial components, calculated by the formula (1). different ratios of initial components, calculated by the formula (1). The addition of the isocyanate and then methacrylate fragments to PLA molecules is confirmed by The addition of the isocyanate and then methacrylate fragments to PLA molecules is confirmed 1H- and 13C-NMR spectra. The 1H-NMR spectra of the initial reactants (PLA, ITI, EGM) is shown in the by 1Н- and 13С-NMR spectra. The 1H-NMR spectra of the initial reactants (PLA, ITI, EGM) is shown Supplementary Materials (Figure S2c). The analysis shows that the 1H-NMR spectrum of a sample of in the Supplementary Materials (Figure S2c). The analysis shows that the 1Н-NMR spectrum of a the isocyanate PLA derivative (Figure 10A) contains only signals from the polylactide chain (a, b) and sample of the isocyanate PLA derivative (Figure 10A) contains only signals from the polylactide ITI (c–h). The reaction is confirmed by the appearance of signals of the “g” urethane group protons near chain (a, b) and ITI (c–h). The reaction is confirmed by the appearance of signals of the “g” urethane 4.73 ppm and by the shift of signals of the “h” protons of the tertiary carbon atom to a stronger field group protons near 4.73 ppm and by the shift of signals of the “h” protons of the tertiary carbon (Figure 10A). With the formation of the methacrylate derivative, new signals appear in addition to the atom to a stronger field (Figure 10A). With the formation of the methacrylate derivative, new earlier observed ones, which belong to the monomethacrylate ester of ethyleneglycol (i–n) (Figure 10B). signals appear in addition to the earlier observed ones, which belong to the monomethacrylate ester A signal appears, which is assigned to the “i” proton of the second urethane fragment, and a shift of of ethyleneglycol (i–n) (Figure 10B). A signal appears, which is assigned to the “i” proton of the the “f” proton signals towards a stronger field also occurs. The addition of monoethyleneglycol ester second urethane fragment, and a shift of the “f” proton signals towards a stronger field also occurs. to the polymer chain of polylactide is also proven by the 13C-NMR spectroscopy data (Figure 10C,D). The addition of monoethyleneglycol ester to the polymer chain of polylactide is also proven by the The appearance of signals in the region of 120–140 ppm in the spectrum of the methacrylate derivative 13С-NMR spectroscopy data (Figure 10C,D). The appearance of signals in the region of 120–140 ppm indicates the methacrylate residue having embedded into the polymer. in the spectrum of the methacrylate derivative indicates the methacrylate residue having embedded into the polymer.

Polymers 2020, 12, 2525 10 of 19 Polymers 2020, 12, x FOR PEER REVIEW 11 of 21

Figure 10. Cont.

Polymers 2020, 12, 2525 11 of 19 Polymers 2020, 12, x FOR PEER REVIEW 12 of 21

FigureFigure 10. 10.(A )(A The) The1H-NMR 1Н-NMR spectrum spectrumof ofthe the PLAPLA isocyanateisocyanate de derivative,rivative, obtained obtained at at the the first ﬁrst stage. stage. (B)( TheB) The1H-NMR 1Н-NMR spectrum spectrum of of the the PLA PLA methacrylate methacrylatederivative, derivative, obtained at the the second second stage. stage. (C (C) The) The 13C-NMR13C-NMR spectrum spectrum of of the the PLA PLA isocyanate isocyanate derivative, derivative, obtainedobtained at the ﬁrstfirst stage. ( (DD)) The The 1313СC-NMR-NMR spectrumspectrum of theof the PLA PLA methacrylate methacrylate derivative, derivative, obtained obtained at at thethe secondsecond stage.

Polymers 2020,, 12,, 2525x FOR PEER REVIEW 1213 of 1921

It should be noted that the presence of the ITI and EGM excess in the reaction mixture leads to side Itreactions should beproceeding noted that with the presencethe formation of the of ITI 2–9% and EGMof oligourethanedimethacrylate excess in the reaction mixture (OUDM), leads toa sideproduct reactions of the proceeding ITI interaction with thewith formation EGM in of the 2–9% ratio of oligourethanedimethacrylateof 1:2 [34]. However, the so-formed (OUDM), aoligourethanedimethacrylates product of the ITI interaction are withshown EGM to be in able the to ratio play of the 1:2 role [34 ].of crosslinking However, the agents so-formed in the oligourethanedimethacrylatesphotopolymerization of modified are PL shownA. Other to be side able products, to play as the well role as of unreacted crosslinking reactants, agents appear in the photopolymerizationnot to hinder the photocuring of modified reactions PLA. Otherand lower side products,the quality as of well the as prepared unreacted scaffolds. reactants, All appear these notcomponents to hinder are the readily photocuring removed reactions from the and prepared lower thecrosslinked quality of structure the prepared during sca itsff washing.olds. All Thus, these componentsthe reaction aremixture readily may removed be used from as thea basis prepared for a crosslinked photoactive structure composition during as its is, washing. without Thus, any thepurification. reaction mixture The following may be usedaddition as a basisof more for OUDM a photoactive and PI composition is only needed as is, for without the preparation any purification. of 3D Thecrosslinked following compositions addition of with more optimal OUDM physicochemical and PI is only needed characteristics. for the preparation All these findings of 3D crosslinked make the compositionsobtained dry withreaction optimal mixture physicochemical very convenient characteristics. for fast preparation All these findings of a photoactive make the obtainedcomposition dry reactionand creation mixture of scaffolds very convenient by laser stereolithography. for fast preparation of a photoactive composition and creation of scaffolds by laser stereolithography. 3.2. The Effect of the Crosslinking Agent (OUDM) Concentration on the Structure of Photocured 3.2.Compositions The Effect of the Crosslinking Agent (OUDM) Concentration on the Structure of Photocured Compositions As mentionedmentioned above, above, oligourethanedimethacrylate, oligourethanedimethacrylate, formed formed as a sideas a productside product at the secondat the stagesecond of synthesisstage of synthesis from the from ITI (remaining the ITI (remaining in the reaction in the mixture reaction after mixture the first after stage the offirst the stage process) of the interaction process) withinteraction EGM, playswith EGM, a role ofplays a crosslinking a role of a agentcrosslinking in the photopolymerization agent in the photopolymerization reaction of methacrylated reaction of PLA.methacrylated At the same PLA. time, At thethe OUDMsame time, content the OUDM in the photoactive content in the composition photoactive appears composition to influence appears the stabilityto influence of the the obtained stability 3D of crosslinkedthe obtained structures. 3D crosslinked Indeed, structures. at the OUDM Indeed, content at the of OUDM less than content 10 wt. %,of noless formation than 10 wt. of a %, stable no crosslinkedformation of structure a stable (insoluble crosslinked in organicstructure solvents) (insoluble takes in place. organic On solvents) the other hand,takes place. the addition On the ofother more hand, than the 17 addition wt. % of of the more crosslinking than 17 wt. agent % of leads the crosslinking to an increase agent of fragilityleads to andan increase the sample’s of fragility destruction. and the The sample’s most stable destruction. 3D-crosslinked The most structures stable 3D-crosslinked are achieved atstructures the 15 wt. are % OUDMachieved content at the in15 thewt. composition. % OUDM content Photoactive in the compositionscomposition. withPhotoactive such content compositions are used with in further such tests.content In are particular, used in based further on tests. such In compositions, particular, based crosslinked on such structures compositions with a, sizecrosslinked of up to structures 3 mm are prepared,with a size which of up areto 3 used mm inare the prepared, cytotoxicity which testing are us aftered in their the storagecytotoxicity in tetrahydrofuran testing after their and storage water (Figurein tetrahydrofuran 11). and water (Figure 11). As we alreadyalready mentioned,mentioned, the application of PLA methacrylate,methacrylate, modifiedmodified via the urethane formation reaction in a solution, in 3D laser stereolithographystereolithography is hindered by partial crosslinking. This resultsresults in in a decreasea decrease of theof opticalthe optical transparency transparency of films of prepared films prepared from the from polymer the and polymer worsening and ofworsening the conditions of the for conditions photoprinting for photoprinting of 3D structures. of 3D At thestructures. same time, At methacrylatedthe same time, PLA methacrylated obtained in thePLA supercritical obtained in carbonthe supercritical dioxide medium carbon isdioxide optically me transparentdium is optically and does transparent not limit and the possibilitiesdoes not limit of laserthe possibilities 3D photoprinting. of laser A typical 3D photoprinting. structure fabricated A bytypical two-photon structure polymerization fabricated fromby two-photon polylactide methacrylatedpolymerization in from the scCOpolylactide2 medium methacrylated is presented in in the Figure scCO 112 medium. is presented in Figure 11.

Figure 11. A 3D crosslinked structure (sca(scaffold),ﬀold), prepared as aa resultresult ofof two-photontwo-photon polymerizationpolymerization of a compositioncomposition consisting of modiﬁedmodified methacrylate-containing PLA, 15 wt. % of OUDM (oligourethanedimethacrylate),(oligourethanedimethacrylate), andand 55 wt.wt. %% ofof thethe photoinitiator.photoinitiator.

Polymers 2020, 12, x FOR PEER REVIEW 14 of 21 Polymers 2020, 12, 2525 13 of 19 3.3. Study of the Local Stiffness of Crosslinked Structures 3.3. StudyThe Young’s of the Local modulus Stiffness (E) of Crosslinkedof scaffolds Structures (measured by nanoindentation) appears to depend not only Theon Young’sthe crosslinker modulus content (E) of scabutff oldsalso (measuredon the photocuring by nanoindentation) technique. appearsIn Figure to 12, depend effective not onlyYoung’s on themoduli crosslinker are presented, content which but alsoare obtained on the photocuring by averaging technique. the corresponding In Figure local 12 ,values effective for Young’ssamples moduliof polylactide are presented, films whichand scaffolds are obtained photocured by averaging by different the corresponding techniques. local The valuesstandard for samplesdeviation of for polylactide each curing films technique and scaff oldsis also photocured displayed, by and diff erenttheir techniques.values are presented The standard in the deviation figure forlegend. each As curing seen technique from Figure is also 12, displayed,the highest and Young’s their values modulus are presentedvalue is reached in the figurefor scaffolds legend. Asfabricated seen from by laser Figure stereolithography. 12, the highest Young’s The dispersion modulus in valuethe values is reached for different for sca samplesffolds fabricated is relatedby to laserthe surface stereolithography. inhomogeneity The and dispersion depends in on the the values technique for diff oferent their samples preparation: is related from to the surfacehighest inhomogeneitydispersion for mercury and depends lamp onirradiation the technique to the oflowest their dispersion preparation: for from laser the stereolithography, highest dispersion which for mercurycorresponds lamp to irradiation the increase to the in lowestthe crosslinking dispersion forhomogeneity. laser stereolithography, Besides, multilayered which corresponds scaffolds to of the a increasecertain shape in the crosslinkingare more conveniently homogeneity. prepared Besides, multilayeredby two-photon sca ffpolymerization.olds of a certain The shape process are more of convenientlytwo-photon preparedabsorption by causing two-photon photopolymerizat polymerization.ion The occurs process in of an two-photon extremely absorption localized causing space photopolymerizationresembling a prolate occursellipsoid, in anwhich extremely is located localized in the space laser resembling beam waist a prolate (in the ellipsoid, experiments, which the is locatedheight inis the~5 laserµm, beamthe diameter waist (in in the the experiments, XY plane theis height~3 µm). is ~5Thisµm, fact the allows diameter the in formation the XY plane of issophisticated ~3 µm). This structures fact allows of the an formation optional ofdesign, sophisticated setting structurestheir density of an by optional variation design, of the setting distance their densitybetween by individual variation passes of the distance of the laser between beam—XY-hatch individual passes (xyh, ofnm). the laser beam—XY-hatch (xyh, nm).

FigureFigure 12.12. Dependence of Young’s modulus of 3D crosslinkedcrosslinked structures (films(films and scascaffolds)ffolds) onon thethe photocuringphotocuring technique.technique. DRT-1000 lamp photocuring of aa film:film: E == 16.9 MPa (standard(standard deviationdeviation (SD) is 8.2 MPa). Scaffolds fabricated by two-photon polymerization (xyh = 4): E = 50.8 MPa (SD (SD) is ±±8.2 MPa). Scaffolds fabricated by two-photon polymerization (xyh = 4): E = 50.8 MPa (SD is is 15 MPa). Scaffolds fabricated by two-photon polymerization (xyh = 2): E = 62.2 MPa (SD is ±15± MPa). Scaffolds fabricated by two-photon polymerization (xyh = 2): E = 62.2 MPa (SD is ±14.3 14.3 MPa). LED photocuring of a film: E = 61.3 MPa (SD is 18.5 MPa, the relative error is 6%). ±MPa). LED photocuring of a film: E = 61.3 MPa (SD is ±18.5 MPa,± the relative error is 6%). Scaffolds Scaffolds fabricated by laser stereolithography: E = 102.5 MPa (SD is 23.2 MPa). The composition of fabricated by laser stereolithography: E = 102.5 MPa (SD is ±23.2 ±MPa). The composition of the themixture mixture for forphotopolymerization photopolymerization is isas asfollows: follows: meth methacrylatedacrylated PLA, PLA, 15 15 wt. wt. % % of of OUDM OUDM and and 5 wt. % ofof thethe PI PI (propidium (propidium iodide). iodide). The The OUDM OUDM and and PI contents PI contents are calculated are calculated withrespect with respect to the PLA to the weight. PLA 3.4. DTAweight. Study of Crosslinked Samples

3.4. DTAThe degreeStudy of of Crosslinked crosslinking Samples of the obtained systems is also estimated based on the coke residue in the TGA analysis of crosslinked PLA methacrylates (Figure 13, Table1). The value of the coke The degree of crosslinking of the obtained systems is also estimated based on the coke residue residue after the polymer destruction reﬂects the degree of the matrix crosslinking since crosslinked in the TGA analysis of crosslinked PLA methacrylates (Figure 13, Table 1). The value of the coke structural fragments are carbonized and form a coke residue at elevated temperatures. According to residue after the polymer destruction reflects the degree of the matrix crosslinking since crosslinked the DTA data (Figure 13), in the destruction of methacrylated PLA cured by laser stereolithography, structural fragments are carbonized and form a coke residue at elevated temperatures. According to the coke residue is 15%, while a LED-photocured sample forms only 9% of the coke. At the same time, the DTA data (Figure 13), in the destruction of methacrylated PLA cured by laser stereolithography, the modiﬁed PLA irradiated by the mercury lamp is completely destructed, without the formation of a the coke residue is 15%, while a LED-photocured sample forms only 9% of the coke. At the same

Polymers 2020, 12, x FOR PEER REVIEW 15 of 21 Polymers 2020, 12, 2525 14 of 19 time, the modified PLA irradiated by the mercury lamp is completely destructed, without the formationcoke residue, of a which coke testiﬁesresidue,the which absence testifies of crosslinked the absence fragments of crosslinked in its structure. fragments Thus, in its the structure. highest Thus,degree the of highest crosslinking degree is observedof crosslinking for samples is observ cureded for by samples laser stereolithography. cured by laser stereolithography.

Figure 13.13. ThermooxidativeThermooxidative destruction destruction curves curves of photocured of photocured methacrylated methacrylated PLA obtained PLA obtained by: 1—laser by: 1—laserstereolithography, stereolithography, 2—LED 2—LED irradiation, irradiation, 3—mercury 3—mer lampcury irradiation. lamp irradiation. The weight The weight loss (TGA, loss (TGA, %) is %)shown is shown by solid by solid lines, lines, and theand rate the ofrate weight of weight loss (W,loss %(W,/min) %/min) is shown is shown by dash-dotted by dash-dotted lines. lines. See See the thephotocuring photocuring conditions conditions in the in Materialsthe Materials and and Methods. Methods.

The above conclusion is confirmed also by the character of the weight loss (TGA) in the The above conclusion is confirmed also by the character of the weight loss (TGA) in the thermooxidative destruction of samples undergone different photocuring procedures. Indeed, as seen thermooxidative destruction of samples undergone different photocuring procedures. Indeed, as seenfrom thefrom TGA the curves TGA (Figurecurves 13 (Figure), the main 13), weight the main loss (∆weightm) of samplesloss (Δ occursm) of insamples the temperature occurs in range the temperatureof 400–500 ◦C, range the highest of 400–500∆m being °С, the observed highest for Δ samplesm being irradiated observed withfor samples the mercury irradiated lamp, whichwith the are mercuryburned almost lamp, completelywhich are burned (curve almost 3). At thecompletely same time, (curve∆m 3). is decreasedAt the same for time, samples Δm thatis decreased undergo forthe samples LED irradiation that undergo (curve the 2), andLED this irradiation stage almost (curve disappears 2), and this in the stage thermooxidative almost disappears destruction in the thermooxidativeof samples obtained destruction by laser stereolithographyof samples obtained (curve by 1)laser with stereolithography the formation of the(curve highest 1) with amount the formationof the coke of residue. the highest This findingamount alsoof the indicates coke residu that structurale. This finding fragments also ofindicates the modified that structural PLA are fragmentscrosslinked of to the the highestmodified degree PLA inare the crosslinked conditions ofto laserthe highest stereolithography degree in thanthe conditions they are during of laser the stereolithographyother procedures. Thethan decrease they are ofduring the rate the of other destruction procedures. when The going decrease from mercury of the rate lamp-photocured of destruction whensamples going to samples from mercury cured by lamp-photocured laser stereolithography sample testifiess to samples this fact, cured as wellby laser (Table stereolithography1). testifies this fact, as well (Table 1). Table 1. The thermooxidative destruction parameters of modified PLA, cured by different techniques.

Table 1. The thermooxidative destruction parameters of modified PLA, cured by% different of Coke No. Technique of Photocuring * ∆m 400–500 ◦C, % WMax,%/min techniques. Residue at 500 ◦C Laser stereolithography 1 Δ6m 400–500 W 0.7мах, % of Coke 15 Residue No. (λ = 263 Technique nm, 0.2 W of/cm Photocuring2) * LED photocuring of a film °С, % %/min at 500 °С 2 11 2.7 9 Laser(λ = stereolithography365 nm, 5 W/cm2) 1 6 0.7 15 DRT-1000 lamp photocuring2 of a film 3 (λ = 263 nm, 0.2 W/cm ) 15 4.4 0 λ 2 LED( = photocuring253 nm, 0.03 of W a/ cmfilm) 2 ≈ 11 2.7 9 * See(λ = the 365 photocuring nm, 5 W/cm conditions2) on the Materials and Methods, where ∆m is the weight loss in the temperature range of 400–500 ◦C, WMax is the highest rate of the weight loss in the temperature range of 400–500 ◦C. DRT-1000 lamp photocuring of a film (λ = 3 15 4.4 0 2 3.5. Cytotoxicity253 nm, ≈0.03 Analysis W/cm ) * See the photocuring conditions on the Materials and Methods, where Δm is the weight loss in the Sca olds and films from the material containing 15 wt. % of OUDM are tested for cytotoxicity temperatureff range of 400–500 °С, Wмах is the highest rate of the weight loss in the temperature range usingof live400–500/dead °С staining. and AlamarBlue and MTT-assays (Figure 14), which are standard for the preliminary assessment of new materials before in vivo experiments [46,47,52–54]. Live/dead staining 3.5.is widely Cytotoxicity applied Analysis and based on two dyes—calcein-AM and ethidium homodimer-1 (propidium iodide),

Polymers 2020, 12, x FOR PEER REVIEW 16 of 21

Scaffolds and films from the material containing 15 wt. % of OUDM are tested for cytotoxicity using live/dead staining and AlamarBlue and MTT-assays (Figure 14), which are standard for the preliminary assessment of new materials before in vivo experiments [46,47,52–54]. Live/dead Polymersstaining2020, 12 is, 2525 widely applied and based on two dyes—calcein-AM and ethidium homodimer-115 of 19 (propidium iodide), which help to visualize the cell state. While penetrating into a viable cell, the whichfirst help dye to indicates visualize the the intracellular cell state. While esterase penetrating activity; intohowever, a viable the cell, second the first one dye can indicatesstain only the dead intracellularcells with esterase the increased activity; membrane however, thepermeability. second one Both can stainMTT onlyand deadAlamarBlue cells with assays the increasedare based on membranemetabolic permeability. reactions that Both occurred MTT and in mitochondria; AlamarBlue assays however, are basedtheir mechanisms on metabolic are reactions different. that In the occurredfirst case, in mitochondria; tetrazolium however,salt is mainly their mechanisms transformed are by di mitochondrialfferent. In the firstsuccinic case, tetrazoliumdehydrogenases salt of is mainlyviable transformed cells; so, water-insoluble by mitochondrial formazan succinic crystals dehydrogenases form. In of the viable second cells; case, so, water-insoluble the reduction of formazanAlamarBlue crystals (resazurin) form. In to the resorufin second case,occurs, the which reduction causes of the AlamarBlue medium color (resazurin) change toand resorufin increase in occurs,fluorescence which causes [55]. the In medium our experiments, color change live/dead and increase staining in fluorescence has shown [55 ].that In our in experiments,72 h after the live/inoculation,dead staining fibroblasts has shown are that attached in 72 h after to the the inoculation,matrix surface, fibroblasts spread are on attached it, andto have the matrixa typical surface,morphology spread on (bipolar it, and haveor multipolar a typical morphology elongated cells) (bipolar; no ordead multipolar or rounded elongated cells are cells); observed no dead (Figure or rounded14A). cells Using are AlamarBlue observed (Figure and 14MTTA). Usingassays, AlamarBlue we revealed and no MTT significant assays, wecytotoxicity revealed no because significant the cell cytotoxicityviability becausefor all the the dilutions cell viability of the formatrix all theextract dilutions is higher of thethan matrix 70% (Figure extract 14B,C). is higher SDS than is used 70% as a (Figurepositive 14B,C). control SDS is to used show as athat positive the used control cell toculture show thatis susceptible the used cell to culturetoxic agents. is susceptible The revealed to toxic data agents.correlates The revealed with datathe correlatespreviously with reported the previously results reportedon other results PLA on othermodifications. PLA modifications. Particularly, Particularly,tetrafunctional tetrafunctional PLA structured PLA structured using two-photon using two-photon polymerization polymerization also alsoshows shows no cytotoxic no cytotoxic effects effectsand and induces induces bone bone formation formation in vivo in vivo [48]. [48 ].

FigureFigure 14. 14.Cytotoxicity Cytotoxicity analysis analysis of aof crosslinked a crosslinked structure structure (sca ff(scaffold)old) obtained obtained by two-photonby two-photon polymerization,polymerization, consisting consisting of the of modified the modified methacrylate-containing methacrylate-containing PLA, 15 PLA, wt. % 15 of OUDM,wt. % of and OUDM, 5% of and a photoinitiator:5% of a photoinitiator: (A)—Live/dead (A)—Live/dead staining (live staining cells—green (live (Calcein cells—green AM), dead(Calcein cells—red AM), dead (propidium cells—red iodide), violet dots—scaffold). Confocal microscopy; (B)—AlamarBlue assay; (C)—MTT assay. SDS is used as a positive control. Polymers 2020, 12, 2525 16 of 19

4. Conclusions We have realized a two-stage modification of polylactide in the supercritical carbon dioxide medium with the introduction of unsaturated methacrylate groups into PLA macromolecules. The optimal ratio of the initial components (based on the corresponding study of the modification efficiency) and the two-stage reaction design in the supercritical fluid medium have allowed obtaining a methacrylate PLA derivative with the degree of modification exceeding 80%. The modified polylactide has been shown suitable for photostructuring using such techniques as photocuring with UV irradiation of a mercury lamp or a LED, 3D printing by stereolithography, or two-photon polymerization. Using the most accurate and promising technique of two-photon polymerization, we have fabricated 3D crosslinked structures (scaffolds) from the modified PLA. The optimal content of the crosslinking agent (OUDM), providing the formation of stable stiff crosslinked structures, is found to be about 15%. The Young’s modulus of the prepared structures depends, among other factors, on the photocuring technique: the structures formed by laser stereolithography have the highest Young’s modulus. The DTA study has shown that Young’s moduli of the structures correlate with the degree of the polymer matrix crosslinking, which depends on the photocuring technique. As a study result, nontoxic and easy-to-handle material has been prepared, with the use of low toxic methacrylate modifying agents and an environmentally friendly solvent, scCO2. The material is capable of structuring with laser additive technologies for application in tissue engineering.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4360/12/11/2525/s1, Figure S1: The initial SEC elution profiles of the re-precipitation-purified product mixture, obtained at the 1 1 second stage, Figure S2a. H-NMR spectrum of the initial polylactide ( H-NMR ((CDCl3) 600 MHz) δ, ppm: 1 1.48–1.66 (m, 3H, CH3), 4.37 (d, 1H, CH), 5.27–5.16 (m, 1H, CH)), Figure S2b. H-NMR spectrum of the initial 1 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate ( H-NMR ((CDCl3) 600 MHz) δ, ppm: 0.99, 1.01 (d, 9H, CH3c,d), 1.05, 1.08 (d, 6H, CH2e), 1.09–1.94 (15H, CH3c,d, CH2e), 3.07 (s, 2H, CH2f), 3.3 (q, 2H, CHf), 3.55, 3.67 (m, 1 1 1H, CHh)), Figure S2c. H-NMR spectrum of the initial ethyleneglycol monomethacrylate ( H-NMR ((CDCl3) 600 MHz) δ, ppm: 1.97 (s, 3H, CH3n), 2.17 (s, 1H, OHp), 3.87 (t, 2H, CH2j), 4.30 (t, 2H, CH2k), 5.61 (s, 1H, CH2l,m), 6.15 (s, 1H, CH2l,m)). Author Contributions: Conceptualization, N.N.G., V.T.S., and A.B.S.; Data curation, I.A.M., N.A.A., and D.S.K.; Funding acquisition, P.S.T. and A.B.S.; Investigation, V.S.K., V.T.S., N.V.M., A.I.S., T.S.Z., B.S.S., and I.V.S.; Methodology, V.S.K., N.N.G., I.A.M., and N.V.M.; Project administration, P.S.T.; Resources, I.A.M. and P.S.T.; Supervision, N.N.G., D.S.K., and A.B.S.; Validation, N.V.M.; Visualization, N.V.M.; Writing—original draft, V.S.K., A.I.S., T.S.Z., and B.S.S.; Writing—review and editing, N.A.A., A.S.K., I.V.S., E.A.B., and A.B.S. All authors have read and agreed to the published version of the manuscript. Funding: This study was conducted in the framework of the Russian Government assignment No. V. 46.14, No. 0082-2014-0006 (AAAA-A17 -117-117032750202-6) (NMR studies), the grant of the Russian Foundation for Basic Research No. 18-29-06019 mk (procedure for the obtaining of methacrylate derivatives of PLA), the grant of the Russian Foundation for Basic Research No. 18-29-06059 mk (cytotoxicity analysis of 3D scaffolds), and Russian Scientific Foundation No. 19-75-10008 (polymer 3D structuring). Acknowledgments: The authors thank Svetlana Kotova (Institute of Chemical Physics, Russia) for a helpful discussion of the manuscript and O. O. Vasilieva for PLA samples handling. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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