**Advances in Thermoresponsive Polymers**

Editor

**Mattia Sponchioni**

MDPI ' Basel ' Beijing ' Wuhan ' Barcelona ' Belgrade ' Manchester ' Tokyo ' Cluj ' Tianjin

*Editor* Mattia Sponchioni Department of Chemistry, Materials and Chemical Engineering "Giulio Natta" Politecnico di Milano Milano Italy

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Polymers* (ISSN 2073-4360) (available at: www.mdpi.com/journal/polymers/special issues/adv thermoresponsive polymer).

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## **Contents**

## **About the Editor**

#### **Mattia Sponchioni**

Mattia Sponchioni received his MSc cum laude in Chemical Engineering in 2015 and his PhD cum laude in Industrial Chemistry and Chemical Engineering in 2018, both at Politecnico di Milano. The focus of his PhD research was on the development of thermoresponsive polymers for biomedical applications. After his PhD, Mattia Sponchioni spent one year as a post-doc at ETH Zurich in 2019, working on the development of continuous countercurrent chromatographic ¨ processes for the purification of biopharmaceuticals. From 2020 to present, he has been an assistant professor at the Department of Chemistry, Materials and Chemical Engineering "Giulio Natta"at Politecnico di Milano. Here, Mattia Sponchioni leads a biomanufacturing group, active in the development of continuous perfusion processes for the production (upstream) of mRNA and bioplastics via fermentation and in the conversion of the traditional single-column batch chromatographic purifications (downstream) into continuous countercurrent processes based on two twin columns. The vision of this group, inspired by the principles of Industry 4.0, is towards process intensification, to be achieved through both continuous processes and integrated manufacturing, as a way not only to increase the productivity but also the sustainability of biomanufacturing. Within this framework, Mattia Sponchioni is a contact for the EU H2020 Marie-Curie innovative training network "Continuous Downstream Processing of Bioproducts".

## **Preface to "Advances in Thermoresponsive Polymers"**

Stimuli-responsive polymers are leading to a breakthrough in many fields, from biomedicine to oil and gas, to advanced separations. The possibility introduced by these materials to dynamically respond to environmental changes, mimicking the complex behavior of naturally occurring macromolecules that regulate fundamental living functions by responding to external stimuli, allows accessing complex applications that could not even be imagined before. Among these materials, thermoresponsive polymers, able to respond to temperature changes with a sharp and often reversible phase separation, are attracting growing attention from the literature, as confirmed by the exponential increase in the scientific publications about this topic in the last twenty years. This is due to the spontaneous occurrence of thermal gradients and to the possibility of applying temperature stimuli quite easily and inexpensively, which makes thermoresponsive polymers extremely appealing in medical and industrial settings. Given the interest towards these materials, in this work entitled "Advances in Thermoresponsive Polymers", the recent advances in the field are reported. The aim is to provide a clear picture of the frontiers reached in the understanding of the mechanistic behavior associated with the temperature-induced phase separation, the influence of the polymer structure in regulating the macroscopic behavior of these materials and the latest applications for which thermoresponsive polymers show great potential.

> **Mattia Sponchioni** *Editor*

## *Article* **Influence of the Polymer Microstructure over the Phase Separation of Thermo-Responsive Nanoparticles**

**Nicolò Manfredini, Marco Tomasoni, Mattia Sponchioni \* and Davide Moscatelli**

Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, via Mancinelli 7, 20131 Milan, Italy<EMAIL_ADDRESS>(N.M<EMAIL_ADDRESS>(M.T<EMAIL_ADDRESS>(D.M.)

**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** Thermo-responsive nanoparticles (NPs), i.e., colloids with a sharp and often reversible phase separation in response to thermal stimuli, are coming to the forefront due to their dynamic behavior, useful in applications ranging from biomedicine to advanced separations and smart optics. What is guiding the macroscopic behavior of these systems above their critical temperature is mainly the microstructure of the polymer chains of which these NPs are comprised. Therefore, a comprehensive understanding of the influence of the polymer properties over the thermal response is highly required to reproducibly target a specific behavior. In this study, we synthesized thermoresponsive NPs with different size, polymeric microstructure and hydrophilic-lipophilic balance (HLB) and investigated the role of these properties over their phase separation. We first synthesized four different thermo-responsive oligomers via Reversible Addition-Fragmentation Chain Transfer (RAFT) Polymerization of poly(ethylene glycol)methyl ether methacrylate. Then, exploiting the RAFT living character, we chain-extended these oligomers with butyl methacrylate obtaining a library of NPs. Finally, we investigated the NP thermo-responsive behavior, their physical state above the cloud point (Tcp) as well as their reversibility once the stimulus is removed. We concluded that the solid content plays a minor role compared to the relative length of the two blocks forming the polymer chains. In particular, the longer the stabilizer, the more favored the formation of a gel. At the same time, the reversibility is mainly achieved at high HLB, independently from the absolute lengths of the block copolymers.

**Keywords:** polymeric nanoparticles; emulsion polymerization; RAFT; thermo-responsive polymers; smart materials; LCST; phase diagram; phase separation

#### **1. Introduction**

Polymeric nanoparticles (NPs) have been extensively studied since the middle of the previous century for their appealing and controllable interfacial and composition properties [1–3]. These features determined their consolidation in several fields, such as coating [4], painting [5], textile [6], or cosmetics [7].

Nowadays, these colloids are mainly produced via free radical emulsion polymerization (FRPe) [8]. This technique allows the synthesis of concentrated (up to 50–60% *w/w*) NP suspensions, commonly referred to as latexes, directly in water, avoiding the use of any organic solvent [9,10]. Although economically viable and brought to a multi-ton scale to satisfy the ever-increasing market demand for polymer latexes, one of the main drawbacks of FRPe is the limited control over the polymer microstructure and hence over the NP physico-chemical properties [11,12].

In the last decades, this limitation was progressively overcome by the advent of controlled radical polymerizations, which gave new lymph to the field [13–15]. In particular, the high control over the polymer chain microstructure achievable with these polymerization techniques paved the way to highly engineered colloids [16–22], contributing to the spreading of the NP technology towards previously unexplored applications.

**Citation:** Manfredini, N.; Tomasoni, M.; Sponchioni, M.; Moscatelli, D. Influence of the Polymer Microstructure over the Phase Separation of Thermo-Responsive Nanoparticles. *Polymers* **2021**, *13*, 1032. https://doi.org/10.3390/ polym13071032

Academic Editors: Alina Sionkowska and Sebastien Lecommandoux

Received: 9 March 2021 Accepted: 23 March 2021 Published: 26 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

In a similar direction, such controlled radical polymerizations were exploited to tune the response of a particularly appealing class of NPs, the so-called stimuli-responsive NPs, i.e., colloids with a sharp and often reversible phase separation behavior in response to external stimuli. Among the different stimuli that can be exploited to trigger such behavior (e.g., pH [23], temperature [24–28], CO2 [29,30], redox potential [31]), the temperature is surely one of the most interesting, promising and versatile. In fact, the possibility of applying thermal stimuli in a controlled way, coupled with the natural occurrence of thermal gradients in living organisms, made thermo-responsive materials attractive for several applications.

In the literature, two opposite behaviors are reported for thermo-responsive polymers. With reference to the phase diagram, namely the diagram of temperature vs. polymer volume fraction, for a solvent/polymer binary mixture, the thermo-responsive polymers can be miscible with the solvent in any proportion below their binodal curve, while phase separating in a polymer-rich phase above it. The minimum of this curve is called lower critical solution temperature (LCST), while each other point is commonly referred to as cloud point (Tcp), since the formation of polymer-rich droplets above this critical temperature often determines the cloudiness of the system. Oppositely, the binodal curve can form a maximum, known as upper critical solution temperature (UCST), with the thermo-responsive polymer being miscible with the solvent above this curve, while phase separating below it [24,32,33]. Despite examples of both kinds of systems have been reported, the easier capability of modulating the Tcp and the almost insensitivity of their phase behavior to environmental properties rendered polymers with a LCST the most studied. These have been successfully investigated in many different applications ranging from pharmaceutical [17,24,34–36] to oil and gas [37] industries.

What clearly appears from these studies is that the peculiar microstructure of the chains constituting the final NPs has a tremendous impact on the macroscopic thermoresponsive behavior of these colloids, including their reversibility (i.e., the capacity to recover the original physico-chemical properties when the stimulus is removed) [38]. At the same time, guidelines for the selection of the polymer chain requisites necessary to access a desired macroscopic behavior are lacking.

In this work, with the aim of rationalizing this concept, we considered NPs constituted by amphiphilic block copolymers comprising a thermo-responsive chain and a hydrophobic portion and systematically investigated the impact of the length of each chain on the final thermo-responsive behavior. In particular, we first synthesized four different thermo-responsive macromolecular chain transfer agents (macro CTAs) via reversible addition-fragmentation chain transfer (RAFT) polymerization. This technique allows to set a priori the average degree of polymerization (*n*) of the oligomers by simply changing the thermo-responsive monomer (in our case, oligo(ethylene glycol)4 methyl ether methacrylate, hereinafter EG4) to chain transfer agent (CTA) molar ratio. The choice of this thermo-responsive monomer instead of the more common N-isopropylacrylamide (NIPAM) was driven by the higher biocompatibility and poor hysteresis of EG4 that contributed to intensify the research towards this compound [17]. Then, exploiting the living nature of the RAFT polymerization, we chain-extended these macro CTAs with butyl methacrylate (BMA) and targeted different chain lengths (*p*). The adoption of the RAFT emulsion polymerization allowed us to obtain the formation of NPs with a lipophilic core and a thermo-responsive shell (nEG4-pBMA) directly in water. These colloids are expected to be stable as long as the temperature is kept below the Tcp. In fact, in this configuration the thermo-responsive chains should be well hydrated providing steric stabilization to the colloids. On the contrary, as soon as the temperature is raised above the Tcp, a sharp coil-to-globule transition of the EG4 moieties is expected. With the thermo-responsive chains collapsing on the NP surface, the colloidal stability is lost, which leads to the NP aggregation, as schematically reported in Scheme 1. For each combination of *n* and *p*, we evaluated the physical state (precipitate or gel in Scheme 1) of the separated phase as well

as the reversibility of these aggregates once the temperature is lowered again below the Tcp and concluded on the factors governing such macroscopic behavior.

**Scheme 1.** Schematic representation of the expected thermo-responsive behavior for NPs stabilized by a thermoresponsive polymer.

#### **2. Materials and Methods**

#### *2.1. Materials*

′

′ ≥ ≥ 4-Cyano-4-(phenyl-carbonothioylthio) pentanoic acid (CPA, MW = 279.38, 99%, Sigma Aldrich, Saint Louis, MO, USA), 2,2′ -azobis(2-methylpropionamidine)dihydrochloride (V-50, MW = 271.19, 98%, Acros Organics, Geel, Belgium), poly(ethylene glycol)methylether methacrylate (EG4, MW = ca300, Sigma Aldrich, Saint Louis, MO, USA), 4-4′ -azobis (4-cyanovaleric acid) (ACVA, MW = 280.28, Sigma Aldrich, Saint Louis, MO, USA), butyl methacrylate (BMA, ≥99%, MW = 142.20, Fluka Chemika, Buchs, Switzerland), diethyl ether (99.7%, MW = 74.12, Sigma Aldrich, Saint Louis, MO, USA), deuterated chloroform (CDCl3, 99.8%, MW = 120.38, Sigma Aldrich, Saint Louis, MO, USA), ethanol (EtOH, MW = 46.07, ≥99.8%, Sigma Aldrich, Saint Louis, MO, USA), and tetrahydrofuran (THF, ≥99.9%, MW = 72.11, Sigma Aldrich, Saint Louis, MO, USA) were used as received.

#### ≥ *2.2. Synthesis of the Thermo-Responsive Macro CTAs*

Four different thermo-responsive macro CTAs were synthesized via RAFT polymerization of EG4 using ACVA as initiator and CPA as CTA. The ACVA to CPA molar ratio (IA) was kept equal to 1/3 for all the syntheses while the EG4 to CPA molar ratio (*n*) was varied in order to obtain different degrees of polymerization (i.e., *n* = 40, 60, 80, and 100). The polymerizations were carried out in ethanol at a monomer concentration equal to 20% *w/w*. Briefly, to synthesize the macro CTA with *n* = 80, hereinafter 80EG4, 8 g of EG4 (26.6 mmol), 0.093 g of CPA (0.33 mmol, i.e., EG4/CPA = 80 mol/mol), and 0.031 g of ACVA (0.11 mmol, i.e., IA = 1/3 mol/mol) were placed in a round bottom flask equipped with a magnetic stirrer. Then, 32.5 g of ethanol were added and the solution was purged with nitrogen for 20 min. The reaction was let occurring at 65 ◦C for 24 h under stirring at 300 rpm. The solution was then purified through three consecutive precipitations in

diethyl ether in order to remove all the impurities and the unreacted monomer. At the end of the purification process, the solution was centrifuged for 10 min at 5000 rpm to separate the two phases and the polymer pellet was recovered and dried inside a vacuum drying oven (VACUCELL) at 35 ◦C overnight. Finally, the polymer was stored at −20 ◦C. Aliquots of the samples were dissolved in deuterated chloroform (CDCl3) at a concentration equal to 14 mg/mL and analyzed via nuclear magnetic resonance (1H-NMR) on a Bruker Ultrashield 400 MHz spectrometer (Bruker, Billerica, MA, USA) with 64 scans (see reaction scheme and spectrum in Figure S1). The monomer conversion and average degree of polymerization, *n*, were calculated according to Equations (S1) and (S2), respectively, and the results are summarized in Table S1.

In order to calculate the number-average molecular weight (*Mn*) and dispersity (*Ð*), the samples were dissolved in tetrahydrofuran (THF) at a concentration of 4 mg/mL, filtered through a 0.45 µm polytetrafluoroethylene (PTFE) membrane and injected in a Jasco LC-2000Plus (Jasco, Easton, MD, USA) gel permeation chromatograph (GPC). The analysis was performed at a flow rate of 1 mL/min and at a temperature equal to 35 ◦C. Three styrene/divinyl benzene columns in series (300 × 8 mm2 , particle size 5 µm, pore size 1000, 105 and 106 A, respectively) were used for the separation and the signal collected with a refractive index (RI) detector. A pre-column (50 × 8 mm2 ) was added before the first column. The instrument calibration was performed with polystyrene standards.

#### *2.3. Synthesis of Amphiphilic Block Copolymers Self-Assembled into NPs*

The thermo-responsive macro CTAs were chain-extended with butyl methacrylate via RAFT emulsion polymerization in order to produce amphiphilic block copolymers selfassembled into NPs. These block copolymers, hereinafter nEG4-pBMA, were synthesized setting the initiator to macro CTA molar ratio to 1/3 and varying the BMA to macro CTA molar ratio (*p*) in order to obtain copolymers with different degrees of polymerization. The polymerization was performed in water at a concentration equal to 20% *w/w*.

For example, in the synthesis of 80EG4-500BMA (i.e., molar ratio between BMA and 80EG4 equal to 500), 1 g of BMA (7 mmol), 0.363 g of 80EG4 (0.015 mmol, i.e., BMA/80EG4 = 500 mol/mol), and 1.27 mg of V-50 (0.005 mmol, i.e., V-50/80EG4 = 1/3 mol/mol) were mixed in 3.64 g of distilled water. The suspension was purged with nitrogen for 20 min at room temperature to avoid side reactions, mixed using a vortex and placed in a pre-heated oil bath at 50 ◦C under magnetic stirring for 24 h.

At the end of the reaction, the NP suspensions were placed in a cellulose membrane (Spectra/Por) with a molecular weight cut-off of 3.5 kDa and were dialyzed against distilled water for 48 h.

The reaction conversion was first evaluated via thermogravimetric analysis performed on an Ohaus MB35 moisture analyzer [6] and then confirmed via 1H-NMR (Figure S2, Equation (S3)) following the same procedure described in Section 2.2. From the 1H-NMR spectrum, the actual *p* was calculated according to Equation (S4). The block copolymer *Mn* and *Ð* (Table S2) were measured via GPC according to the procedure described in Section 2.2.

The NP size (reported in terms of Z-average, indicated as Dn) and polydispersity index (PDI) were measured via dynamic light scattering (DLS) on a Zetasizer Nano ZS (Malvern, UK) at a scattering angle of 173◦ (Table 1). Each sample was diluted before the analysis to a concentration equal to 1% *w/w* and analyzed in triplicate with 13 runs per measurement.

**Table 1.** Size (Dn), polydispersity index (PDI) and temperature of cloud point (Tcp) of the NP synthesized.

The block copolymer hydrophilic-lipophilic balance (HLB, Table 1) was calculated according to Equation (1):

$$HLB = 20 \frac{M\_h}{M\_t} \tag{1}$$

where *Mh* is the molecular mass of the hydrophilic portion of the molecule (i.e., the thermoresponsive portion in our case), and *Mt* is the molecular mass of the whole molecule. This value is a number comprised within 0–20 with a value of 0 corresponding to a completely lipophilic molecule and a value of 20 corresponding to a completely hydrophilic molecule.

#### *2.4. Study of the Thermo-Responsive Properties*

The NP cloud point (Tcp) was calculated via DLS analyzing the inflection points of Dn vs. temperature curves [17]. The temperature was ramped from 25 ◦C up to 70 ◦C at intervals of 1 ◦C, leaving 120 s of equilibration time before each measurement.

In order to evaluate the NP reversibility, all the samples were diluted at 20%, 10%, 5%, and 1% *w/w* and placed in a pre-heated oven at 80 ◦C for 1 h. At the end of the heating procedure, the NPs were removed and left to equilibrate at room temperature for an additional hour.

The samples showing a reversibility (i.e., turned again cloudy) were analyzed via DLS in order to compare their actual size with the original one (i.e., before the heating procedure).

#### **3. Results and Discussion**

Thermo-responsive amphiphilic block copolymers with a well-defined microstructure and self-assembled into NPs were synthesized via two consecutive RAFT polymerizations.

We first synthesized four thermo-responsive macro CTAs (nEG4, with *n* equal to 40, 60, 80, and 100, respectively) through RAFT solution polymerization of EG4 in ethanol. High monomer conversion (>94%), poor *Ð* (<1.2) and controllable *Mn* were obtained (see Table S1 in the Supporting Information). In fact, one peculiarity of the RAFT process is the possibility of setting *a priori* the desired molecular weight by simply changing the monomer to CTA molar ratio (*n*) as shown in Equation (2) [39].

$$M\_{\rm H\_{\rm mCTA}} = M\_{\rm CTA} + n X M\_{\rm EC\_4} \tag{2}$$

being *X* the monomer conversion, *Mnm*CTA , *M*CPA and *M*EG4 the molecular weights of the produced macro CTA (i.e., number-average molecular weight), CTA and EG4 respectively. This Equation is validated by the linear increase in *Mn* reported in Figure 1a for different values of *n*. At the same time, the RAFT polymerization ensures that the actual *n* of the product, as measured through 1H-NMR, closely matches the target value, as shown in the comparative plot reported in Figure S3a, thus granting a good control over the polymer average chain length.

ಲ

ಲ = ் + ாீర

**Figure 1.** (**a**) Number average molecular weight as function of the degree of polymerization of the thermo-responsive block (**b**) Tcp as a function of the degree of polymerization of the thermo-responsive block (**c**) Normalized chromatograms of the block copolymers synthesized from 100EG4 having p equal to 0 (black), 275 (light blue), 600 (grey), 850 (red), and 1200 (blue), respectively. ir represents the normalized intensity of the refractive index detector. (**d**) Number-average molecular weight as a function of the degree of polymerization of the lipophilic block for polymers having *n* equal to 40 (square), 60 (circle), 80 (triangle), and 100 (star), respectively.

It is worth noticing that this increase in the polymer molecular weight is not followed by a change in its Tcp, which changed in a very narrow range between 62 and 66 ◦C, as demonstrated in Figure 1b. The phase separation at this temperature may find application in the oil and gas filed, where thermo-responsive polymers can be applied as viscosity modifiers or to enhance the emulsification/separation of oil in water during the so-called enhanced oil recovery [37,40,41]. However, we would like to point out that with this work we are not aiming at a particular application. Rather, EG4 was selected as a representative of the oligo(ethylene glycol)methyl ether methacrylates, with a controllable phase separation. The Tcp of these polymers can be readily adjusted to the desired value by choosing the appropriate oligo(ethylene glycol) chain length or by copolymerization with hydrophilic (i.e., Tcp increases) or hydrophobic (i.e., Tcp decreases) monomers [24]. The poor sensitivity

of the Tcp to the molecular weight for this class of polymers, quite expected for LCST-type polymers at least for these low values of *n*, allows to obtain a portfolio of macro CTAs with comparable Tcp and different *Mn*.

These macro CTAs were then chain-extended with BMA via RAFT emulsion polymerization to obtain amphiphilic block copolymers self-assembled into NPs. Different degrees of polymerization for the polyBMA portion (*p*) were targeted. Again, the RAFT polymerization ensured to achieve values of *p* very close to the target (Figure S3b). In addition, all the copolymers synthesized presented high monomer conversion (>95%) and poor *Ð* (<1.4, Table S2) as expected from a controlled radical polymerization. The possibility of forming well-defined block copolymers is further demonstrated by the high blocking efficiency. In fact, in most of the samples, the macro CTA showed high conversion during the polymerization as its characteristic peak is strongly reduced in intensity in the GPC chromatogram of the block copolymers. This is shown as an example in Figure 1c for the 100EG4-*p*BMA samples. The peak of the macro CTA at 24.5 min is seen as a shoulder in the chromatogram of the 100EG4-1200BMA, while it is not separated and resolved in all the other samples, suggesting high conversion. Indeed, for these samples, low molecular weight tails that could hide the signal from the macro CTA were recorded. However, their intensity is quite small compared to that of the main copolymer distribution, suggesting that in the worst scenario the residual macro CTA concentration is low as well.

This two-step RAFT polymerization strategy provides two degrees of freedom (*n* and *p*) enabling to independently control the partition of the thermo-responsive and hydrophobic units and the copolymer *Mn,* according to Equation (3) (Figure 1d):

$$M\_{\rm nl} = M\_{\rm nl\_{\rm mCTA}} + pXM\_{\rm BMA} = M\_{\rm CTA} + nM\_{\rm EG\_4} + pXM\_{\rm BMA} \tag{3}$$

with *M*BMA and *Mn* being the BMA and final block-copolymer number-average molecular weight, respectively. In particular, through the tuning of *n* and *p* it is possible to produce copolymers with the same *Mn* and different hydrophilic-to-lipophilic balance (HLB). To rationalize this concept, we calculated for all the samples (Table 1) the corresponding HLB (Equation (1)), which was found to play a relevant role in dictating the macroscopic thermo-responsive behavior, as discussed in the following. The HLB could be varied in the range 1–10 and, therefore, block copolymers from very hydrophobic to slightly hydrophilic could be produced with this approach.

Another advantage of using the RAFT emulsion polymerization is the simultaneous synthesis of the copolymer and its self-assembly into NPs at high solid content. The high control in the block copolymer synthesis allows the formation of colloids with tunable size (Dn) and poor polydispersity indexes (PDI) as clearly shown in Table 1.

In particular, the two-step approach adopted in this work makes the NP size a function of *n* and *p* (Equation (4)) [39], thus providing the opportunity of decoupling Dn from the polymer molecular weight and in turn of obtaining NPs with comparable size but different microstructure:

$$D\_n = \frac{6 \text{ } p \text{ } M\_{\text{BMA}}}{A \text{ } N \text{ } \rho} = \frac{6 \text{ } p \text{ } M\_{\text{BMA}}}{(\alpha + n\beta) \text{ } N \text{ } \rho} \tag{4}$$

with *M*BMA being the molecular weight of BMA, *ρ* the density of the lipophilic chains, *N* the Avogadro number, *A* the area on the NP surface covered by a single polymeric chain, *α* and *β* two constants characteristic of the polymer. In particular, *A* is a function of the molecular weight of the hydrophilic portion of the block copolymer when amphiphilic stabilizers as *n*EG4 are used at a concentration higher than their critical micellar concentration (CMC). In this case, the lipophilic backbone is expected to dispose tangentially to the NP surface with the hydrophilic moieties exposed to the bulk. In a similar conformation, the higher *n* (and in turn *Mn* of the stabilizer), the higher the portion of the surface that it is able to cover and, in turn, the lower the average NP size [39]. The NP synthesized in this work are in line with this prediction and their size, at given *p*, decreases with *n* as clearly visible in Figure 2a. In fact, the higher *n* the higher the surface area covered by a single chain with a

consequent reduction in the NP size (Equation (4)). Moreover, a linear increase in the NP size with *p*, at a given *n*, is obtained independently from the stabilizer used (Figure 2a).

=

6 ெ =

ெ

6 ெ ሺ + ሻ

**Figure 2.** (**a**) NP size (Dn) as a function of *p* in the case of *n* equal to 40 (square), 60 (circle), 80 (triangle), and 100 (star), respectively. (**b**) NP size as function of external temperature in the case of 80EG4 -650BMA.

It is worth mentioning that a broad range of Dn can be accessed with this strategy, from around 50 nm up to 1 µm (i.e., 40EG4-3000BMA). This is a remarkable result for colloids produced via RAFT emulsion polymerization, which typically leads to NPs with an upper size limit of 500 nm [42].

The high control achievable with this two-step RAFT polymerization strategy allows the formation of a wide portfolio of colloids with different size and HLB suitable for a systematic analysis of the impact of these parameters over the macroscopic thermoresponsive behavior.

First, we measured the Tcp of the different samples via DLS, according to the procedure described in Section 2.4. All the NPs show a similar trend with a sharp change in their size from nm to few µm once the Tcp is reached (Figure 2b). It is worth mentioning that the absolute value of the size reached above the Tcp should be only considered as an indication, since at this size scale the gravity causes the sedimentation of the larger aggregates already within the time of the analysis. What is reliable is the dynamic of the phase separation, which is due to the loss of steric stability. In fact, once the Tcp is overcome, the thermo-responsive shell collapses on the NP core leading to aggregation (Scheme 1). It is worth noticing that the Tcp is independent from *p* for all the *n* considered (Table 1). In fact, the high conversions obtained in the synthesis of the stabilizer coupled with the high compartmentalization achievable with the RAFT emulsion polymerization permits to obtain a library of NPs with a transition temperature that is dependent only on the thermo-responsive monomer chosen [17–25].

After having demonstrated that all of the samples exhibit a thermo-responsive behavior, their physical state above the Tcp as well as their reversibility once the thermal stimulus is removed were systematically investigated and related to the three degrees of freedom characterizing the system, i.e., solid content, *n* and *p*.

We explored the thermo-responsive behavior at four different concentrations, namely 1, 5, 10, and 20% *w/w* according to the procedure described in Section 2.4. Interestingly, no significant effects of NP concentration over their reversibility were noticed for all of the NPs analyzed, as visible in Figure S4. Thus, samples with a reversible transition at given *n* and *p*, are so at any of the concentrations tested. Moreover, only a modest effect on the NP physical state above the Tcp was observed with a tendency to the formation of gel when the concentration is increased. Then, the effect of *n* and *p* was investigated in details at a fixed concentration of 20% *w/w*. We divided the phase diagram into four classes depending on the NP reversibility as well as the physical state of the aggregate above the Tcp. In particular, we individuated two possible physical states, namely precipitate and gel (Figure S5). In the former, the destabilization led to the NP aggregation and precipitation of a bulky polymer phase, while in the case of the gel, the destabilization involved the whole mass of the sample, leading to a self-standing monolith. Both of these states can be reversible or irreversible depending on the capability of the NPs to recover their original size once the thermal stimulus is removed, as shown for example in Figure S6 for the sample 60EG4-150BMA. The four different behaviors are indicated in the phase diagram of Figure 3a as reversible precipitate, irreversible precipitate, irreversible gel, and reversible gel, respectively. In general, we observed that an increase in *p* always led to a progressive loss in the reversibility of the phase separation, independently from the *n* considered. This behavior can be justified considering that an increase in *p* is associated with an increase in the NP lipophilicity, leading to stronger hydrophobic interactions between the aggregated NPs. This hypothesis is supported by the evidence that by counter-balancing the increase in lipophilicity of the block copolymer with an increase in the thermo-responsive portion (i.e., *n*), the range of *p* leading to reversible transitions is expanded. In addition, *n* seems to play a relevant role in the physical state of the phase separated system, favoring the formation of hydrogels. This may be attributed to the decrease in the NP size, and then to an increase in their number, associated with an increase in *n*, when the solid content is fixed. This higher number of particles, together with the partial shell interpenetration could lead to a percolating system, in which at a critical NP distance the system stops flowing forming a self-standing gel [43].

Since, as shown, a change in *p* and *n* is always accomplished with a variation in the NP size making the understanding of the real impact of these parameters difficult, we reanalyzed the samples as a function of their HLB (Equation (1)). Interestingly, it was found that NPs with comparable HLB present similar size (Figure S7). Moreover, the decreasing trend of Dn with the HLB observed for all the *n* considered is in line with Equation (4). Grouping NPs with similar size allows to understand the impact of the absolute length of both blocks on the NP reversibility. In fact, as visible from Figure 3b, for HLB lower than 4.5, an irreversible aggregation is obtained independently from the *n* considered. However, the systems stabilized by shorter *n* (40 and 60 in Table S1) are more prone to the reversibility (achieved at lower HLB compared to the samples with *n* = 80 and 100). This result suggests that the longer the stabilizer, the lower the NP reversibility when size and lipophilicity are fixed, which may be due to a certain interaction between the thermo-responsive shells. Hence, a fine tuning of the block copolymer microstructure allows to decouple the NP size and HLB from their thermo-responsive response.

**Figure 3.** (**a**) Phase diagram reporting the physical state of the NP suspension above its Tcp at a fixed concentration of 20% *w/w* being RP, reversible precipitate, IP, irreversible precipitate, IG, irreversible gel, and RG, reversible gel, respectively. (**b**) Phase diagram reporting the reversibility of the phase separation at a given concentration of 20% *w/w* and as a function of the HLB of the block copolymer and *n*.

#### **4. Conclusions**

In this work, we correlated the macroscopic behavior of thermo-responsive NPs with the microstructure of the block copolymer they are made up of. The use of two sequential RAFT polymerization steps allows the synthesis of colloids with high blocking efficiency and controllable properties including size, molecular weight, HLB and microstructure. Moreover, exploiting the two degrees of freedom that this procedure allows, it was possible to decouple all the NP properties in order to better clarify the role of each parameter over the final macroscopic behavior.

First, it was found that the solid content does not play a significative role on the properties of these systems. In general, a decrease in the nanoparticle size is accomplished with a better reversibility of the system and a major tendency to form gel. However, this behavior is impacted also from the absolute length of both the portions (*n* and *p*) of the block copolymers that form the NP. In fact, NPs with comparable HLB and size recover their reversibility more easily when the stabilizing block is shorter.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/polym13071032/s1, Figure S1: 1H-NMR spectrum of 60EG4 ; Figure S2: 1H-NMR spectrum of 60EG4 -400BMA; Figure S3: (a) thermo-responsive polymer degree of polymerization calculated via 1H-NMR (*n*NMR) as a function of the EG4/CTA mole ratio (*n*target). (b) hydrophobic block degree of polymerization calculated via 1H-NMR (pNMR) as function of the BMA/macro CTA mole ratio (ptarget); Figure S4: Phase diagram reporting the reversibility of the phase separation as a function of the NP concentration and p in the case of *n* equal to 40 (a), 60 (b), 80 (c) and 100 (d), being RP = Reversible Precipitate, IP = Irreversible Precipitate, IG = Irreversible Gel and RG = Reversible Gel, respectively; Figure S5: NP forming a precipitate (left) or a hydrogel (right) once the temperature is increased above the Tcp; Figure S6: NP size as function of temperature during heating (triangle) and cooling (star) in the case of 60EG4 -150BMA; Figure S7: NP size as function of block copolymers HLB in the case of *n* equal to 40 (square), 60 (circle), 80 (triangle) and 100 (star); Table S1: conversion (X), degree of polymerization (*n*), number-average molecular weight (Mn) and dispersity (Ð) of the macro CTAs synthesized; Table S2: conversion (X), degree of polymerization (*n*, *p*), number-average molecular weight (Mn) and dispersity (Ð) of the block copolymers synthesized; Equation (S1): EG4 conversion; Equation (S2): Degree of polymerization for the macro CTA from the corresponding 1H-NMR spectrum; Equation (S3): BMA conversion; Equation (S4): Degree of polymerization for the polyBMA from the corresponding 1H-NMR spectrum.

**Author Contributions:** Funding acquisition: D.M.; investigation: N.M.; methodology: M.S.; supervision: D.M.; validation: M.T.; writing—original draft: N.M.; writing—review and editing: M.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors are grateful to Roberto Panebianco for the help with the experimental part.

**Conflicts of Interest:** The authors declare no conflict of interests for this publication.

#### **References**

## *Article* **Inhomogeneities in PNIPAM Aqueous Solutions: The Inside View by Spin Probe EPR Spectroscopy**

**Ekaterina M. Zubanova 1, \* , Sergei V. Kostjuk 2,3,4 , Peter S. Timashev 1,3,5 , Yury A. Rochev 3,6 , Alexander I. Kokorin 5,7 , Mikhail Ya. Melnikov 1 and Elena N. Golubeva 1**

**Abstract:** Coil to globule transition in poly(N-isopropylacrylamide) aqueous solutions was studied using spin probe continuous-wave electronic paramagnetic resonance (CW EPR) spectroscopy with an amphiphilic TEMPO radical as a guest molecule. Using Cu(II) ions as the "quencher" for fastmoving radicals in the liquid phase allowed obtaining the individual spectra of TEMPO radicals in polymer globule and observing inhomogeneities in solutions before globule collapsing. EPR spectra simulations confirm the formation of molten globules at the first step with further collapsing and water molecules coming out of the globule, making it denser.

**Keywords:** thermoresponsive polymers; electronic paramagnetic resonance; spin probe; nitroxides; coil to globule

#### **1. Introduction**

Thermoresponsive polymers are of great interest because they undergo coil-to-globule transitions of single-polymer chains in polar solvents near the lower critical solution temperature (LCST) [1–3]. This peculiarity gives them potential for biomedical and pharmaceutical applications such as drug and gene delivery, tissue engineering, cell expansion, sensors, microarrays, and imaging [4–9]. Phase transition in the thermoresponsive polymers solutions passes due to molecular interaction and the cohesion of solvent molecules with hydrophilic fragments in the polymer chains [10,11]. Hydrophilic groups form hydrogen bonds with water molecules at low temperatures, resulting in good solubility in aqueous solutions. Increasing the temperature leads to the degradation of the hydrogen bonds system and the formation of an intramolecular interaction between polymers chains, further collapsing the polymer globule. Poly(N-isopropylacrylamide) (PNIPAM) is one of the most studied thermoresponsive polymers, which has LCST in a physiologically relevant temperature range of ≈32 ◦C in aqueous solutions [1]. Macroscopic methods (turbidimetry, DSC, etc.) usually fix sharp and reversible changes of PNIPAM properties in the vicinity of LCST [11,12]. In addition, the formation of small, even nanoscopic inhomogeneities of polymer gels or films in different solvents before LCST is proved by continuous-wave electron paramagnetic resonance spectroscopy (CW EPR) [13–15]. The EPR spectra of paramagnetic molecules (spin probes) are sensitive to the microenvironment and can give valuable information concerning collapse processes at the molecular level [14,16,17]. Stable

**Citation:** Zubanova, E.M.; Kostjuk, S.V.; Timashev, P.S.; Rochev, Y.A.; Kokorin, A.I.; Melnikov, M.Y.; Golubeva, E.N. Inhomogeneities in PNIPAM Aqueous Solutions: The Inside View by Spin Probe EPR Spectroscopy. *Polymers* **2021**, *13*, 3829. https://doi.org/10.3390/ polym13213829

Academic Editor: Mattia Sponchioni

Received: 9 October 2021 Accepted: 2 November 2021 Published: 5 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

nitroxide radicals containing a >N–O•·group have been the most popular spin probes during the last 55 years [17,18]. Hyperfine interaction of the unpaired electron spin with the magnetic moment of 14N (S = 1) nucleus leads to splitting of the EPR signal of nitroxides to three lines. Local polarity, viscosity, and the ability of a media to form hydrogen bonds influence the electron density distribution in the spin probe, affecting the shape of the EPR signal and spin-Hamiltonian parameters (g-tensor and hyperfine splitting (hfs) tensor), which can be estimated by modeling the EPR spectra. The small amphiphilic radical 2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) (ca. 6.7 Å in diameter) is a powerful tool to detect and control the formation of polymer globules [14,19–21]. It is known that the micropolarity in polymeric globules of thermoresponsive polymers is significantly lower than the polarity of water and is close to that of chloroform [18]. A lower polarity and higher viscosity of the globules [15] result in changes of spin Hamiltonian parameters and the line widths of TEMPO spectra compared with those in aqueous solution, reflecting a decrease of the amplitudes of the spectral lines belonging to the radicals in the solution and the appearance of broader components of the TEMPO spectrum corresponding to probes localized in the globules. This effect was used by Junk et al. [19] to reveal the formation of heterogeneities in thin photocrosslinked films of PNIPAM notably earlier and later the LCST detected by macroscopic methods. However, the spin probe technique was not applied to study coil-to-globule transitions and the nature of inhomogeneities in PNIPAM aqueous solutions up to now. In the present paper, we applied CW EPR to study the structure and features of formation of nano- and/or micro heterogeneities of PNIPAM with two different polydispersities and the dynamics of spin probes inside the globules and in aqueous solutions upon heating from room temperature.

#### **2. Materials and Methods**

#### *2.1. Substances*

The stable radical (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) and copper(II) chloride dihydrate CuCl2·2H2O purchased from Sigma-Aldrich were used without further purification. N-Isopropylacrylamide (NIPAM) (99%, Acros, Geel, Belgium) was recrystallized from solution in *n*-hexane, dried in vacuum, and then stored under argon atmosphere. 2,2′ -Azobis(2-methylpropionitrile) (AIBN) (98%, Sigma-Aldrich, Burlington, MA, USA) was recrystallized from ethanol and dried in vacuum at 20 ◦C. Benzene (anhydrous, 99.8%, Sigma-Aldrich, USA) and *n*-hexane (reagent grade, Ekos-1) were used as received.

#### *2.2. Polymer Synthesis*

Two poly(N-isopropylacrylamide) (PNIPAM) samples I and II were synthesized via conventional radical and RAFT polymerization, respectively. The sample I (numberaverage molar mass Mn = 175.5 kDa, polydispersity index Ð = 4.3; yield: 97%) was prepared by free radical polymerization in benzene at 60 ◦C for 24 h using azobisisobutyronitrile (AIBN) as an initiator according to the procedure reported in [20]. Then, the reaction mixture was precipitated in n-hexane. Then, the obtained polymer was purified by dissolving in acetone followed by precipitation in n-hexane at least three times, and the product was dried at 45 ◦C in a vacuum oven. Sample II (Mn = 107.6 kDa, Ð = 2.05; yield: 95%) was prepared by RAFT-mediated radical polymerization. The polymerization was carried out in benzene at 60 ◦C for 24 h using 2-(dodecylthiocarbonothioylthio)-2 methylpropionic acid as the RAFT agent. The polymerization was conducted in an argon atmosphere in a Schlenk reactor equipped with a magnetic stir bar. The reactor was charged by N-isopropylacrylamide (0.502 g, 4.44 mmol), vacuumed, and filled with argon. Then, 0.22 mL of 0.02 M benzene solution of RAF-agent, 1.11 mM of 0.02 M benzene solution of AIBN, and 2.87 mL of benzene were added into the reactor. The mixture was bubbled with argon for 30 min, and then, the reactor was placed into an oil bath heated to 60 ◦C. After 24 h, the reactor was opened and frozen with liquid nitrogen. The polymer purification was performed similarly to the purification of sample I (see above).

#### *2.3. Polymers Characterization*

#### 2.3.1. H NMR Spectroscopy

1H NMR (500 MHz) spectra were recorded in CDCl3 at 25 ◦C on a Bruker AC-500 (Bruker, Karlsruhe, Germany) spectrometer calibrated relative to the residual solvent resonance. The 1H NMR spectra of sample I and sample II are presented in Figure A1 (see Appendix A).

#### 2.3.2. Spectrophotometry

The temperature transition was monitored by UV-Vis spectrophotometry using a Victor Nivo instrument (Perkin Elmer, Waltham, MA, USA) at a wavelength of 405 nm. The studied polymer solution was poured into a 96 (48)-well plate, and the absorbance at each point was registered. The range of measurement was 4–40 ◦C. The cloud points were determined at 10% of the transmission reduction.

#### 2.3.3. Differential Scanning Calorimetry (DSC)

The DSC studies were carried out using a NETZSCH STA 449 F3 synchronous thermal analyzer (Selb, Germany) in a helium atmosphere at gas flow rates of 70 mL/min (main) and 50 mL/min (protective). An aluminum crucible was used with a solution weight of 30–45 mg. The calibration of the temperature and sensitivity of the device was carried out using standard samples of adamantane, indium, and distilled water. The aqueous solution of the polymer sample (5 wt %) was heated and cooled at 2 K/min. TGA measurements were performed with the same device using solid PNIPAM samples at heating rate of 20 ◦C min−1 under nitrogen flow.

#### 2.3.4. Size Exclusion Chromatography

Size exclusion chromatography was performed using an Ultimate 3000 Thermo Scientific chromatographic complex (Thermo Fisher Scientific, Waltham, MA, USA) equipped with PLgel precolumn guard (Agilent, Santa Clara, CA, USA, size 7.5 × 50 mm, particle size 5 µm) and PLgel MIXED-C column (Agilent, size 7.5 × 300 mm, particle size 5 µm) thermostated at 50 ◦C. The elution was performed in the isocratic mode with DMF containing 0.10 M LiBr at a flow rate of 1.0 mL min−1 . SEC traces were recorded as mentioned above, and polymethylmethacrylate standards (ReadyCal Kit, PSS GmbH) with Mw/Mn ≤ 1.05 were used to calculate Mw/Mn.

#### *2.4. EPR Samples Preparation*

In all cases, ≈0.5 mM TEMPO and 10 wt % PNIPAM freshwater solutions were prepared, dissolving the required predefined amounts of TEMPO radical and PNIPAM polymer. In the first step, the dissolution of TEMPO was performed at room temperature using ultrasonication. Then, PNIPAM was added, and the mixture was aged at 4 ◦C for 24 h until the complete dissolution of the polymer. The solutions were put into glass tubes with 2 mm inner diameter; then, the tubes were sealed to prevent water evaporation.

#### *2.5. EPR Measurements*

EPR spectra were recorded using X-band spectrometer Bruker EMX-500 (Bruker, Karlsruhe, Germany). The temperature of the samples was varied in the range of 300–353 K using the flow of nitrogen gas. The thermostatic device from Bruker was used; the accuracy of the temperature setting was about ±0.5 K. Each sample was left at the particular temperature for precisely 5 min for equilibration before recording. At 305 K, the waiting time was 60 min to equilibrate the samples. Typical parameters of the spectra recording were a microwave power of 0.8 mW, modulation amplitude of 0.04 mT, and a sweep width of 8 mT. «Quenching» of fast spin probes in water was performed by adding 10 mg of CuCl2·2H2O to 0.5 mL PNIPAM solution, as recommended in [21].

#### *2.6. Data Analysis*

The integration of EPR spectra and the amplitude measuring were performed by the EsrD program developed at the Chemistry Department of Lomonosov Moscow State University [16]. For LCST measurements, the amplitude value of TEMPO EPR spectra was normalized to the Q-factor [22] at each temperature and to the amplitude of the left component of the spectra at 300 K. All spectra simulations were performed using homemade scripts for the MATLAB program employing an Easyspin (v. 5.2.28) toolbox [23]. Spectra of TEMPO radical in solutions (type *A*) were simulated using a model based on fast motion implemented as a 'garlic' function in Easyspin. Simulations of the spectra of radicals in polymer globule (type *B*) were made in a slow-motion regime using a 'chili' function from the Easyspin program. The slow-motion model 'chili' is based on the Schneider–Freed theory [24], solving equations for slow tumbling nitroxides. Anisotropic values of Spin-Hamiltonian parameters (*g*-tensor and hyperfine splitting (hfs) tensor, usually denoted as *a*-tensor) were averaged to obtain *g*iso and *a*iso values, the rotational correlation time tensor (*τcorr*), denoted as the average time required for the rotation of a molecule at one radian, was calculated from averaged rotational diffusion constant. Fitting errors for *g*iso and *a*siso were ±0.00003 and ±0.01 mT, respectively. More details of the spectra simulation are presented in Appendix A.

#### **3. Results**

According to DSC (Figure A2 in Appendix A) data from the heating step, the measured LCST of the polymers I and II in aqueous solutions was ≈305 K and did not depend on the synthesis method and molar mass distribution of polymers, which is typical for PNIPAM solutions [25]. TGA measurements showed that both PNIPAM samples are stable at the temperatures used in this work (≤353 K), and the thermal decomposition (5 wt % weight loss) began at ≈625 K (Figure A3a). Note that the weight loss between 320 and 400 K (Figure A3a) is consistent with the presence of ca. 10–12 wt % of water in poly(N-isopropylacrylamide) samples. This is confirmed by the disappearance of this weight loss after annealing the samples at 470 K for 15 min (Figure A3b).

At temperatures below LSCT (305 K), the EPR spectrum of TEMPO in PNIPAM solution (sample I, 10 wt%) and TEMPO spectrum in water have a shape close to the isotropic fast-motion limit (Figure 1). The EPR spectrum of TEMPO in the polymer II solution and its changes during heating were very similar to those of polymer I. However, all these spectra (see Figure A4 in Appendix A) are slightly asymmetric; i.e., the signal amplitude above the baseline is bigger than that below it. This asymmetry may appear due to the following reasons. Firstly, the existence of the solvent shells with different polarities around TEMPO molecules in aqueous media revealed by the analysis of *Q*band EPR spectra was postulated by Hunold et al. [26]. This fact leads to a bimodal distribution of magnetic and dynamic parameters of TEMPO probes, which manifest itself in asymmetric signals due to the superposition of the spectra. Secondly, the asymmetry of the nitroxide signal can be caused either by the intermolecular spin-exchange interaction between spin probes in solutions [27,28] or by mixing with the dispersion signal of the resonance effect [29]. Fortunately, in our case, the observed effects are too small and can be neglected to simplify the spectra modeling in aqueous solutions. According to our simulation, the TEMPO spectrum in water at room temperature corresponds to probes with a *g*iso value equal to 2.00579 and the hfs constant *a*iso equal to 1.73 mT. The isotropic rotational correlation time (*τcorr*) is about 11 ps. TEMPO in 10 wt % PNIPAM solution has parameters typical for less polar media: *g*iso equal to 2.00585 and *a*iso is equal to 1.72 mT, wherein *τcorr* = 16 ps, manifesting the higher viscosity of polymer solution. A simple estimation of viscosity using the Stocks–Einstein equation from rotational correlation time shows increasing viscosity of 10 wt% PNIPAM solution below LCST in ≈1.5 times compared to pure water.

**Figure 1.** Normalized EPR spectra of TEMPO radical in 10 wt% PNIPAM aqueous solutions at 300–353 K registered upon heating.

At temperatures higher than 305 K, the intensity of the TEMPO spectra decreases rapidly, and the lines shape changes: the spectra become broader, and the central line turns to more asymmetric. At 320 K and higher, the components in the high-field region corresponding to the probe in the hydrophobic globule clearly manifest themselves in the spectra. Changes in the lines' shape can be empirically presented as the amplitude A of the high field component vs. temperature T (Figure 2). As seen from Figure 2, this dependence allows measuring the LCST of polymer solutions. A fast drop of the amplitude occurs due to the appearing of the more broadened and hence less intensive signal of TEMPO radicals located in globules.

At temperatures above the LCST, the TEMPO spectrum in the PNIPAM solution appears to be the sum of at least two signals. One of them belongs to the radicals in the solution (type *A*). At the same time, the position and shape of the second signal (denoted as the spectrum of *B*-type probes) manifest a more hydrophobic local environment and a hindered rotation of TEMPO radicals comparing to *A* probes. The simulation of experimental spectra as the sum of the spectra of two probes applies a considerable number of parameters, the variation of which leads to similar changes in EPR spectra. For example,

a shift of the components can occur both due to changes in the hfs constant or rotational correlation time. The use of the spin-Hamiltonian and dynamic parameters of radicals *A* at different temperatures determined from the temperature dependence of TEMPO spectra in an aqueous solution makes it possible to slightly reduce the number of parameters to be varied in the modeling. Nevertheless, the parameters of probes *B* cannot be quantitatively determined from the simulation of experimental spectra due to their simultaneous influence on the shape of the spectral line and overlapping signals of probes *B* with the component corresponding to the hyperfine splitting on 13C nuclei of TEMPO, the so-called satellite in the probes *A* spectrum. The spectrum of *B* probes can be elucidated by subtracting the spectrum of centers *A* from the total spectrum or as the spectra of polymer films swollen in the probe solution [19]. Nevertheless, the subtracting does not allow obtaining a fully "pure" single-particle spectrum and may lead to artifacts in the line shape.

**Figure 2.** The normalized amplitude of the high-field component of TEMPO EPR signal in PNIPAM solution (green squares correspond to polymer I, red circles correspond to polymer II) vs. temperature.

The perspective method to get the individual spectrum of radicals inside a polymer globule is "quenching" the signal of rapidly rotating radicals *A* due to the spin-exchange broadening of EPR lines [21]. A Heisenberg exchange interaction occurs between unpaired electrons as a result of the collision of two paramagnetic particles, leading to an exchange of spin states between partners. In turn, the exchange of spin states between partners leads to a change in the width of the EPR line of the paramagnetic particle up to its confluence with the baseline. Such broadening may appear in the presence of some inert paramagnetic particles, e.g., Cu(II) ions, not chemically interacting with components of solutions. Figure A5 presents the change of the EPR spectra of TEMPO radicals in aqueous solutions in the presence of Cu(II) ions. Adding small amounts of the paramagnetic ions firstly leads to minor broadening of the signal of motile radicals in the solution. Further increasing of Cu(II) concentration continues to broaden the TEMPO signal up to the baseline. The optimal concentration of Cu(II) ions is about 0.2 M, and this amount was used for "quenching" the signal of radicals *A* in PNIPAM (polymer I) solutions.

Due to the spin exchange between Cu(II) ions and TEMPO, the experimental spectrum transfers practically to a base line at the temperature range of 295–301 K. At 302 K, a new slight signal of slow-moving radicals appears, and its intensity and double integral increase with temperature rising (see Figure A6, Appendix A). We suppose that the new signal corresponds to TEMPO radicals located in inhomogeneities of the polymer solution and not contacted with the quencher. Over 305 K, polymer globules start collapsing, and the spectra become more intensive due to an increase in the fraction of the radicals captured by polymer globules. Heating up to 323 K leads to a further increase in the TEMPO signal, but the lines' shape does not change noticeably. At 333–353 K, the components of the spectra narrow, apparently, because of the increasing mobility of TEMPO radicals. The number of spin probes that depends linearly on the double integral of EPR spectra stays constant at 333–353 K. We assume that the spectrum at 353 K shown in Figure 3 matches the TEMPO spectra of the radicals in dense PNIPAM globules (probes *B*). According to our simulation results, these spectra correspond to the anisotropic rotation of TEMPO radicals with *g*iso and *a*iso equal to 2.00615 and 1.60 mT, respectively. Such value of the isotropic hfs constant matches to the local polarity close to that of the chloroform or low-molecular alcohols [30]. Rotational movements of radicals are anisotropic and more hindered along the *x*-axis: *D*x = 7.6 × 106 s −1 < *D*z = 8.7 × 107 s −1 < *D*y = 5.9 × 108 s −1 , where *D*x,y,z are the components of the rotational diffusion tensor. The obtained magnetic and dynamic parameters of TEMPO radicals in PNIPAM globules (probes B) were used in our simulations of the total spectra series (Figure 1). − − −

**Figure 3.** EPR spectrum of TEMPO radical in PNIPAM solution at 353 K in the presence of Cu(II) ions. Blue line—experimental spectrum, red dots—simulated spectrum.

All spectra of the samples I and II at temperatures above 305 K were excellently fitted as a sum of the spectra of two types: "hydrophilic, fast" *A* probes and "hydrophobic, slow" *B* probes (Appendix A, Figure A7). The root-mean-square deviation obtained from simulations of all fitted spectra was less than 1%. The complete simulation results obtained from EPR spectra fitting at several temperatures are collected in Appendix A in Table A1. The difference between the two samples comes out only as the content of the *B* probes at 305 K: 26% in sample I and 44% in sample II. This difference may occur due to various polydispersities of polymers I (*Ð* = 4.3) and II (*Ð* = 2.05). Actually, the content of macromolecules with similar molecular mass is bigger in polymer II, leading to a more rapid globule collapse and the capturing of bigger content of the TEMPO radical. The simulation results of the EPR spectra of samples I and II registered higher than 305 K are similar, so only the simulation results obtained for sample II will be discussed further.

Figure 4 illustrates the fitted EPR spectrum recorded at 333 K as a superposition of two individual spectra. The spectrum of probes A looks like a "fast limit" spectrum of TEMPO radicals dissolved in water. Its EPR parameters become less polar with heating due to decreasing the polarity of liquids (for example, the dielectric constant ε of water changes from 78 to 60 at temperatures 298 and 350 K, respectively) [31]. A hyperfine coupling constant also decreases from 1.73 to 1.71 mT, and giso increases from 2.00588 to 2.00598. The rotation correlation time of A probes decreases with heating from 10 to 1 ps due to the diminishing water viscosity [32]. The magnetic and dynamic parameters of type A probes after LCST do not depend on PNIPAM concentration and are similar to those for the pure aqueous solution without PNIPAM.

*Đ Đ*

ε

**Figure 4.** Decomposition of fitted EPR spectrum (red) of TEMPO radical in 10 wt % PNIPAM solution at 333 K into two signals: type *A* radicals (green) and type *B* radicals (blue).

The spectrum of *B* probes (radicals located in hydrophobic globules) is more broadened and looks like a «slow-motion» signal. The EPR spectrum of type *B* at 353 K coincides with the experimental spectrum of the TEMPO radical in the polymer globule obtained by the "quenching" experiment (Figure 3).

Figure 5 shows changes of *a*iso and *g*iso of radicals *A* and *B* as a function of temperature. The complete simulation results obtained from EPR spectra fitting at several temperatures are collected in Table A1 of Appendix A. At 305–313K, the *g*iso values corresponding to *B* particles are similar to those of probe *A* in aqueous solution, but after 313 K, they rise to 2.00615 at 353 K. In contrast, *a*iso values corresponding to *B* probes do not change at 305–353 K being equal to 1.60 mT. The average rotation correlation time of particles *B* is 1300 ps at 305–318 K. After 318 K, a two-fold decrease of *τcorr* to 620 ps resulting in narrowing of the line width of the EPR signal of the *B* probes is observed, signaling that TEMPO molecules in the globule become more mobile. The mole fraction of type *B* probes rises upon heating to 353 K to 70%.

**Figure 5.** Change of *a*iso (**a**) and *g*iso (**b**) of TEMPO radical in PNIPAM solution with heating according to EPR spectra simulation. Green squares and triangles correspond to *A* particles, blue ones correspond to *B* particles.

#### **4. Discussion**

All the applied methods: DSC, spectrophotometry, and the EPR spin probe technique determine the same value of LCST (305 K) in aqueous PNIPAM solutions of polymers I and II, which is consistent with the literature data [33]. Coil-to-globule transition on PNIPAM aqueous solutions studied by the EPR spin probe method occurs in a narrow temperature range of 8–10 K. Thus, in solutions, hydrophilic bonds between water molecules and PNIPAM individual chains quickly degrade, and the rapid formation of polymer globules takes place. To the contrary, Junk et al. also fixed LCST at 305 K in PNIPAM-based photocrosslinked films swollen in water, but the temperature range of the collapse as measured by spin-probe CW EPR spectroscopy was substantially broadened to 40–50 K [19]. Therefore, only one type of radical (*A* probes) prevails in the aqueous solution of PNI-PAM below LSCT (305 K). Nevertheless, at 302–304 K, some part of TEMPO molecules is captured into polymer globules, transferring to *B* probes and testifying to the generation of heterogeneities that begin to collapse at 305 K, when the coil-to-globule transition takes place.

*τ*

● We suppose that the observed increase in g-factor values of *B* particles in the globules with increasing temperature occurs due to the breakdown of hydrogen bonds between water molecules and the N–O• group of TEMPO [34,35]. At higher temperatures, these complexes decompose, and *g*iso rises to lower polar values corresponding to individual TEMPO radicals. In addition, water molecules may come out of the globules while heating, making the media surrounding TEMPO radicals more hydrophobic. Such evolution from molten globule to tight globule was previously observed by Wang et al. [36] by laser scattering for PNIPAM with narrow mass distribution, and it may also lead to the change of *g*iso value to be less polar.

χ χ χ The changes of content in *A* probes obtained from spectra simulation are similar to the normalized amplitude changes of the high-field component of the full TEMPO spectrum in PNIPAM solutions (see Figure 2 and Table A1). Therefore, the amplitude changes may be used not only for LCST determination but for estimation of the content of probe molecules *A*(χA) in the aqueous solution after the globule collapse. Since χA+ χB = 1, the molar fraction of *B* probes could be valued, too. The content of probes in the collapsed globules relates to an affinity of the probe to the polymer and amphiphilicity of the collapsed globules. Thus, the measuring of amplitudes of EPR spectra of probe molecules in thermoresponsive polymers aqueous solutions gives the opportunity to determine and predict its properties using the spin-probe technique without making challenging spectra simulations.

#### **5. Conclusions**

The CW EPR method with the spin-probe technique and "quenching" approach was applied for studying coil-to-globule phase transition in PNIPAM aqueous solution upon heating at 298–353 K. The broadening of fast-moving radicals in water by spin exchange with Cu(II) ions allows obtaining individual spectra of the spin probe in polymer globule after collapsing and reliable spin-Hamiltonian parameters of both types of TEMPO probes in solutions. In addition, the "quenching" approach has shown the formation of inhomogeneities in PNIPAM aqueous solution at 2–3 degrees below the critical temperature, whereas the temperature range of the collapse in PNIPAM-based photocrosslinked films swollen in water is substantially broadened to 40–50 K. A simple analysis based on amplitude measurement of TEMPO EPR spectra registered at different temperatures gives a cloud point and allows estimating the content of probe in collapsed globule. According to EPR spectra simulations, the formation of dense globules at high temperatures through molten ones at the first step takes place in PNIPAM solutions.

**Author Contributions:** Conceptualization, E.N.G. and Y.A.R., investigation and formal analysis, E.M.Z. and S.V.K., writing—original draft preparation, E.M.Z., E.N.G. and S.V.K., writing—review and editing, A.I.K., Y.A.R., P.S.T., project administration, P.S.T. and M.Y.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Russian Foundation for Basic Research (Grant 20-02-00712).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** This work was partially performed using MSU equipment provided by the M.V. Lomonosov Moscow State University Program of Development. The authors are grateful to A.V. Bogdanov for useful discussions and Evgenii Ksendzov for his help in the synthesis and characterization of poly(N-isopropylacrylamide) samples.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Appendix A**

#### *EPR Spectra Simulation Details*

All CW EPR spectra simulations were performed using homemade scripts for MAT-LAB program employing the Easyspin (v. 5.2.28) toolbox [23]. Quality of fitting was controlled by the calculation of root-mean-square deviation for the difference spectra. Spectral and dynamic parameters were obtained by fitting simulated EPR spectra to experimental data using least-squares fitting algorithms.

The spectra of TEMPO radical (type *A*) in solutions were simulated using a model based on fast-motion implemented as a 'garlic' function in Easyspin. Line broadening was described by isotropic rotation correlation time, and by additional convolutional line broadening caused mostly by unresolved hyperfine splitting between unpaired electron and nuclear spin (usually 1H). As initial values for the diagonal elements of the g-tensor and hyperfine splitting tensor on 14N nuclei for type *A* probes, the following values were used:

$$\mathbf{g\_{x}} = \text{2.0092}, \mathbf{g\_{y}} = \text{2.0062}, \mathbf{g\_{z}} = \text{2.0022};$$

$$\mathbf{A\_{\infty}} = 0.71 \text{ mT}, \mathbf{A\_{YY}} = 0.70 \text{ mT}, \mathbf{A\_{ZZ}} = 3.71 \text{ mT}.$$

In addition to splitting on 14N nuclei in the case of type *A* probes, isotropic hyperfine splitting on 13C isotopes of six CH3-groups with *a*iso = 0.54 mT was considered. To simplify the modeling procedure, we change abundances for carbon isotopes instead of adding six paramagnetic nuclei.

Simulations of the spectra of radicals in polymer globule (type *B*) were made in a slow-motion regime using the 'chili' function from the Easyspin program. The slow-motion model 'chili' is based on the Schneider–Freed theory [24], solving equations for slowtumbling nitroxides. Line broadening was described by the anisotropic rotation correlation time and additional Gaussian line broadening. To start fitting, the following diagonal values of g-tensor and hyperfine interaction a-tensor were used:

gx = 2.0098, gy = 2.0062, gz = 2.0022;

**Figure A1.** 1H NMR spectra of (**a**) sample I and (**b**) sample II. The traces of solvents are marked by asterisks.

$$
\mathbf{2}
$$

**Figure A2.** DSC thermograms of 5 wt % aqueous solutions of poly(N-isopropylacrylamide): (**a**) sample I and (**b**) sample II.

Temperature (o C) **Figure A3.** TGA curves of (**a**) sample I and sample II studies in this work and (**b**) sample II before and after annealing at 470 R for 15 min.

The initial values of magnetic parameters were taken from [37] and corrected using experimental isotropic *g*iso and *a*iso of TEMPO radical in different solvents [30]. During the simulation, only gx and Azz components were varied, being more sensitive to the environment. Isotropic giso and aiso were calculated as an average value of diagonal elements:

C)

heating cycle

$$\mathbf{g\_{iso}} = \frac{1}{3} \left( \mathbf{g\_x} + \mathbf{g\_y} + \mathbf{g\_z} \right)$$

$$a\_{\rm iso} = \frac{1}{3} \left( \mathbf{A\_{xx}} + \mathbf{A\_{yy}} + \mathbf{A\_{zz}} \right)$$

The average rotational correlation time *τcorr* was calculated using the following equations from diagonal elements of diffusion tensor Dr = [*Dxx Dyy Dzz*]:

$$\mathrm{D\_r} = \frac{1}{6\pi\_{corr}}$$

$$\pi\_{\mathrm{corr}} = \frac{1}{6\sqrt[3]{D\_{xxD\_{yyDzz}}}}$$

g

a

ଵ ଷ

*τ*

Dr =

*τ* =

ଵ ଷ g

A

ଵ ఛ

ଵ ටೣೣವವ య

**Figure A4.** EPR spectra of TEMPO radical in water (0.3 mM) (**a**) and 10% PNIPAM solution (0.5 mM) (**b**) at 298 K. Arrows show amplitude value of spectra above and below the baseline. Red dot line shows baseline.

**Figure A5.** EPR spectra of TEMPO radical in water in the presence of Cu(II) ions: (**a**)—[Cu(II)] = 0, [TEMPO] = 0.5 mM; (**b**)—[Cu(II) = 0.003 M, [TEMPO] = 0.33 mM; (**c**)—[Cu(II)] = 0.01 M, [TEMPO] = 0.25 mM; (**d**)—[Cu(II)] = 0.1 M, [TEMPO] = 0.5 mM; (**e**)—[Cu(II)] = 0.2 M, [TEMPO] = 0.5 mM; (**f**)—[Cu(II)] = 0.3 M, [TEMPO] = 0.5 mM. Spectra (**a**–**c**) were divided by 15 for better representation and comparison with (**d**–**f**).

**Figure A6.** Change of double integral of EPR spectra of TEMPO in PNIPAM aqueous solution in the presence of Cu(II) vs. temperature.

**Figure A7.** Simulation of spectra of system TEMPO/PNIPAM/H2O at different temperatures. Blue lines—experimental spectra, red circles—simulation spectra.

*τ τ*

**χ χ**

**Table A1.** Selected spectral parameters from simulation TEMPO EPR spectra in 10 wt% aqueous solutions at 300–353 K.

#### **References**

## *Article* **Characterization of Thermoresponsive Poly-N-Vinylcaprolactam Polymers for Biological Applications**

**Lorenzo Marsili 1,2, \*, Michele Dal Bo 1 , Giorgio Eisele 3 , Ivan Donati 4 , Federico Berti 2,† and Giuseppe Toffoli 1,†**

**Abstract:** Poly-N-Vinylcaprolactam (PNVCL) is a thermoresponsive polymer that exhibits lower critical solution temperature (LCST) between 25 and 50 ◦C. Due to its alleged biocompatibility, this polymer is becoming popular for biomedical and environmental applications. PNVCL with carboxyl terminations has been widely used for the preparation of thermoresponsive copolymers, micro- and nanogels for drug delivery and oncological therapies. However, the fabrication of such specific targeting devices needs standardized and reproducible preparation methods. This requires a deep understanding of how the miscibility behavior of the polymer is affected by its structural properties and the solution environment. In this work, PNVCL-COOH polymers were prepared via free radical polymerization (FRP) in order to exhibit LCST between 33 and 42 ◦C. The structural properties were investigated with NMR, FT-IR and conductimetric titration and the LCST was calculated via UV-VIS and DLS. The LCST is influenced by the molecular mass, as shown by both DLS and viscosimetric values. Finally, the behavior of the polymer was described as function of its concentration and in presence of different biologically relevant environments, such as aqueous buffers, NaCl solutions and human plasma.

**Keywords:** poly-N-vinylcaprolactam; thermoresponsive polymers; LCST

#### **1. Introduction**

Thermoresponsive polymers are characterized by a drastic and discontinuous change of their physical properties with temperature. Given a solvent/polymer binary mixture, the phase diagram usually exhibits a binodal curve that divides a polymer-rich zone from a zone in which the polymer and the solvent are miscible in every proportion. Accordingly, the critical solution temperature can correspond to the minimum or the maximum of the binodal curve. If the critical point corresponds to the minimum of the curve, the corresponding temperature is called lower critical solution temperature (LCST). On the contrary, the temperature corresponding to the maximum of the curve is called upper solution temperature (UCST) [1].

The ability to respond to a change in temperature makes thermoresponsive polymers a "smart" class of materials that can be applied in a broad range of applications [2]. To date, there are hundreds of thermoresponsive polymers developed for various applications in the biological field, which include tissue engineering, bioseparation, drug and gene delivery [2–4]. As a general rule, LCST-type polymers are easily solvated in water through hydrogen bonding and polar interactions [5]. Accordingly, their biological interest relies on

**Citation:** Marsili, L.; Dal Bo, M.; Eisele, G.; Donati, I.; Berti, F.; Toffoli, G. Characterization of Thermoresponsive Poly-N-Vinylcaprolactam Polymers for Biological Applications. *Polymers* **2021**, *13*, 2639. https://doi.org/ 10.3390/polym13162639

Academic Editors: Mattia Sponchioni and Florian J. Stadler

Received: 16 July 2021 Accepted: 4 August 2021 Published: 8 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the presence of a desirable sharp transition in aqueous systems [2]. Their utilization in the biological field requires that the LCST in aqueous solution is close to the normal human physiological temperature, which is usually comprehended between 36.5 and 37.5 ◦C, but can oscillate in a wider range between 33.2 and 38.2 ◦C [6].

PNVCL is a thermoresponsive polymer which exhibits LCST that has been applied in biomedical and environmental applications, in cosmetics and as an anticlogging agent in pipelines [3,4]. PNVCL is usually described as a biocompatible alternative to poly-Nisopropylamide (PNIPAM) [3,4,7–15] as both polymers can exhibit LCST behavior close to the physiological temperature. According to this misconception, PNVCL has been often described as a polymer with a well-defined LCST at 32 ◦C [7–9,16–21], even though the LCST has been observed in broad range of temperature between 25 and 50 ◦C [3,4]. As a matter of fact, PNVCL and PNIPAM exhibit a complete opposite miscibility behavior [3,22]. PNIPAM exhibits a LCST at about 32 ◦C, which is almost independent from the molecular weight (Type II) [3,4]. On the contrary, PNVCL miscibility can be described by the Flory–Huggins theory and depends on molecular weight [3,23], polymer, salt and protein concentration (Type I). Due to its molecular weight-dependent LCST, the control of both molecular weight and polydispersity in PNVCL synthesis is of crucial importance [3,4,22–27] for any application. Similarly, the determination of molecular mass must rely on appropriate methodologies that allow to correlate the synthetic procedures with the final properties of the polymer.

Although the thermoresponsivity of PNVCL was first observed in 1968 [23], the polymer is still introduced as a novelty in biological and nanoscientific publications [3,4,7–9,16–21]. The first "in vitro" evaluation of PNVCL cytotoxicity on intestinal Caco-2 and pulmonary Calu-3 cell lines was published in 2005. The test demonstrated great cell tolerance although PNVCL exhibited toxic effects above its LCST [28]. To date, the biocompatibility of PNVCL has been established in several different cell lines, including different types of human carcinomas [4,29–31]. PNVCL was also investigated as a suitable environment for cell proliferation and manipulation [32] and it is commercialized as a hair setting product under the name of Luviscol® Plus [33]. The utilization of PNVCL with carboxyl end groups has been reported for the preparation of PNVCL-based delivery systems in conjunction with biocompatible polysaccharides, such as alginates [34], chitosan [7,9,16–21,35], or dextran [3,36,37]. A common approach is to use PNVCL-COOH polymers with a 32 ◦C LCST as starting materials for the preparation of biocompatible copolymers and thermoresponsive particles [7–9,19,35,38]. The LCST is raised according to the degree of substitution and the molecular mass of the polymer conjugated with PNVCL. However, to ensure reproducibility, PNVCL synthesis must be optimized in order to ensure control over chemical structure, molecular mass, polydispersity and morphology [3,4]. Moreover, it is still fundamental to assess the polymer thermoresponsive behavior in biologically relevant fluids such as human plasma.

The aim of this work is to rationalize this concept, as we considered the polymerization of different PNVCL polymers with carboxyl terminations (PNVCL-COOH) that exhibit different LCSTs according to their different molecular mass, their concentration and the solution environment. Furthermore, this work provides insight on different approaches for the determination of the molecular mass and the LCST of PNVCL. The polymers were prepared by using a modified approach of the protocol reported by Prabaharan [35] and reported by many different authors [7–9,16–19]. Accordingly, we reported the preparation of PNVCL-COOH polymers exhibiting LCST between 33 and 42 ◦C via FRP. The hypothesis of mass-dependent LCST was first supported by the comparison of 13C NMR spectra of different PNVCL-COOH, by assessing the variation of the signals related to the terminations as a function of the NVCL/AIBN ratio that were used for the synthesis. Finally, the determination of the molecular mass via viscosimetry and DLS demonstrated that the LCST of PNVCL-COOH is inversely proportional to the molecular mass. LCSTs were determined using UV-VIS and DLS at a concentration of 0.5 wt.%. The variation of LCST of PNVCL-COOH polymers was also assessed in relation to polymer concentration, the

presence of NaCl and three different buffers: citrate (pH 3), acetate (pH 5) and phosphate (pH 7). Finally, we report a method for the evaluation of the polymer in human plasma. In particular, it was observed that it is possible to increase LCST by decreasing PNVCL-COOH concentration, while the presence of salts, particularly phosphates, results in a significant lowering of the LCST. The measurements performed in human plasma showed a relevant lowering of the LCST of PNVCL-COOH polymers by about 10 ◦C. These observations are particularly useful for physiological applications, as the polymers undergoing LCST may result in possible cytotoxic effects.

#### **2. Materials and Methods**

#### *2.1. Materials*

N-Vinyl Caprolactam (NVCL), 2,2 ′ -Azobis(2-methylpropionitrile) (AIBN), 3-mercaptopropionic acid (MPA), were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-hydroxy succinimide (NHS), Deuterium oxide (D2O), N,N′ -Dimethylformamide anhydrous (DMF), Dimethyl sulfoxide-d6, Deuterium Chloride 37% (DCl/D2O) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Spectra/Por TM Cellulose membrane tubing (1–2 kDa, MWCO) was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

#### *2.2. Synthesis of PNVCL-COOH Polymers*

PNVCL-COOH was synthesized by free radical polymerization by using a modified version of the protocol reported by Prabaharan [35]. Prior to their utilization, NVCL was recrystallized in hexane and AIBN was recrystallized in ethanol, while MPA was transferred in a sealed vial that was deoxygenated with Ar. For all preparations, 5 mg of AIBN (0.304 mmol) and 28 µL of MPA (3.278 mmol) were used. Different molar ratios between NVCL and AIBN were used in order to obtain polymer with different molecular mass and LCST. The NVCL/AIBN molar ratio were, respectively, 122, 244, 305, 610, 1220 and 1690. DMF was deoxygenized with Ar for 15 min prior to the addition of the reagents (Figure 1). After the dissolution of the reagents, the reaction was carried out in sealed vials at 70 ◦C for 8 h. All sealed vials were dried overnight at 80 ◦C before the reaction. After the reaction, the solution was dialyzed in a cellulose membrane tubing (MWCO of 1–2 kDa) against distilled water for at least 2 days to remove impurities and unreacted materials. Finally, the frozen product was freeze-dried at −50 ◦C and 0.05 mbar and stored at 4 ◦C.

**Figure 1.** Synthesis of PNVCL-COOH polymers.

#### *2.3. Structural Characterization of PNVCL-COOH Polymers*

NMR spectra were acquired using a High-resolution 500 MHz Bruker NEOn500 Quadruple resonance (H/C/N/2H) equipped with a high-sensitivity TCI 5 mm CryoProbe (Bruker, Billerica, MA, USA) at 25 ◦C in D2O and d6-DMSO.

FT-IR spectra were recorded with a double-beam Perkin Elmer System 2000 Ft-IR Spectrometer (Perkin Elmer, Waltham, MA, USA) in the range of 4500–370 cm−1 using KBr pellets.

The number of terminal carboxyl groups was determined via conductimetric titration. The polymers were dissolved using diluted HCl and the solutions were titrated using a standardized solution of NaOH 0.1 M. The deprotonation of the -COOH end groups resulted in the formation of a small plateau in the conductimetric titration curve. The

number of carboxyl groups was determined by the volume difference of added NaOH solution between the initial and the final point of the plateau (Figure S10).

#### *2.4. UV-VIS Determination of LCST*

Phase transition and absorbance measurements were carried out in a Shimazdu UV-visible spectrophotometer model UV-2450 equipped with a TCC-240A Thermoelectrically Temperature Controlled Cell Holder (Shimazdu, Kyoto, JP). Transmission data were used for the realization of the miscibility curves. The miscibility curves were fitted with a sigmoidal model in order to calculate the LCST as the inflection point by using the following equation:

$$y = \mathcal{T}r\_{\max} + \frac{(\mathcal{T}r\_{\max} - \mathcal{T}r\_{\min})}{1 + e^{\frac{(T - LCST)}{dT}}} \tag{1}$$

where *Tr* is the calculated transmittance at a specific wavelength at a fixed temperature.

#### *2.5. DLS Determination of LCST*

The LCSTs of PNVCL-COOH polymers were determined using a Malvern Zetasizer Nano ZS90 (Malvern Panalytical S.R.L., Malvern, UK). Samples (at a concentration of 0.5 wt.%) were incubated at different temperatures for 5 min in the temperature range between 25 and 50 ◦C and the cuvettes were examined to check visible turbidity. The LCST was attributed to the temperature at which a dramatic change in the shape of the autocorrelation curve was observed [39].

#### *2.6. Determination of Molecular Mass*

#### 2.6.1. Size Exclusion Chromatography

Samples were analyzed using a Viscotek TDA 302 (HP-SEC-TDA, Viscotek, USA), using degassed 0.3 M acetate buffer (pH = 8.0) as solvent. A volume of 100 µL of sample was injected, flow rate was maintained at 0.6 mL/min and the column and detectors temperature were kept at 25 ◦C. Before injection, polymer solutions were filtered through a 0.45 µm cellulose nitrate disposable membrane and the eluent was filtrated through a 16–40 µm glass filter to ensure a low light scattering noise level. A polyethyleneoxide standard (*MW* = 22,411, [*η*] = 0.384 dL/g, *Mw/Mn* = 1.03) was used to normalize the viscometer and the light scattering detectors.

#### 2.6.2. Dynamic Light Scattering

The molecular mass of the polymers was estimated using a Malvern Zetasizer Nano ZS90 (Malvern Panalytical S.R.L., Malvern, UK). All measurements were performed at 25 ◦C at a concentration of 0.5 wt.% in milliQ water. Each sample was analyzed three times and provided a measurement of an average hydrodynamic diameter (*Dh* ) corresponding to the position of a peak in size distribution located between 5 and 30 nm. *Dh* standard deviation is referred to the position of the peaks and does not provide any information on peak width. For a random coil conformation, the average radius of gyration was calculated as *Rg* = *Dh* × 0.75. The average molecular mass was estimated from *Rg* using the equation reported by Lau [40] (Equation (2)) and Eisele [41] (Equation (3)), respectively:

$$R\_{\mathcal{S}} = 2.94 \times 10^{-2} M\_w^{\
u \text{ } 0.54} \tag{2}$$

$$
\left\langle s^2 \right\rangle = 1.77 \times 10^{-18} M\_w^{-1.15} \tag{3}
$$

where h*s* 2 i is the mean square radius of gyration in cm−1 .

#### 2.6.3. Intrinsic Viscosity Measurements

The intrinsic viscosity [*η*] of PNVCL-COOH was measured at 25 ◦C by means of a Schott Geräte AVS/G automatic measuring apparatus and a Ubbelhode capillary viscometer, using water as a solvent. Polymer solutions and solvents were filtered prior to

the analysis through 0.45 µm nitrocellulose filters (Millipore, Germany). [*η*] was calculated from the polymer concentration dependence of the reduced specific viscosity, *ηsp*/c, according to the Huggins Equation (4):

$$
\eta\_{\rm red} = \frac{\eta\_{\rm sp}}{c} = \left[\eta\right] + k[\eta]^2 \mathbf{c} \tag{4}
$$

where *k* is Huggins constant. The values of intrinsic viscosity [*η*] was calculated at infinite dilution by using two calibration lines per sample, one of which was obtained by excluding the values for the most diluted sample. [*η*] was calculated as an average of the values calculated by applying both equations, in order to provide a reasonable confidence interval for the calculated value. The corresponding average viscosimetric molecular weight (*Mη*) of PNVCL-COOH was calculated in agreement with the Mark–Houwink–Sakurada (MHS) Equation (5).

$$\mathbf{h}[\eta] = \mathbf{K} \cdot \mathbf{M}\_{\eta}^{a} \tag{5}$$

*K* and *a* parameters used for the calculation are reported by Kirsh [12,42] for the calculation of the molecular mass in different size range at 25 ◦C. The MHS equations that were used were, respectively:

$$\left[\eta\right] = 35 \times 10^{-3} M\_{\eta}^{0.57} \tag{6}$$

$$\mathbf{a}[\eta] = 38.9 \times 10^{-3} M\_{\eta}^{0.69} \tag{7}$$

#### **3. Results and Discussion**

PNVCL-COOH polymers were prepared via free radical polymerization as reported by Prabaharan [35]. The initial results ruled out the possibility that PNVCL-COOH was able to precipitate in diethyl ether in the conditions described by the original procedure. Accordingly, the procedure was modified in order to produce PNVCL-COOH polymers with higher molecular weight. In free radical polymerization, the degree of polymerization *Xn* is directly proportional to the square of the concentration of the monomer, according to the following equation:

$$\overline{X}\_{\rm{ll}} = \alpha \frac{\left(k\_p\right)^2}{2k\_t v\_p} [M]^2 \tag{8}$$

Consequently, the preparation of PNVCL-COOH with higher molecular weight was achieved by changing the molar ratio between initiator and monomer (M/I). For this reason, PNVCL-COOH were distinguished according to the M/I molar ratio that was used for their synthesis. The M/I was, respectively, 122 (equal to that reported by Prabaharan), 244, 305, 610, 1220 and 1690. The precipitation in diethyl ether was achieved from values of M/I above 610. The elongation of the hydrophobic portion resulted in a different hydrophilichydrophobic balance that allowed the precipitation of PNVCL\_610, PNVCL\_1220 and PNVCL\_1690 in diethyl ether. FRP of PNVCL is associated with a lack of control on polymer polydispersity. This could be related to the low yield of the synthesis after diethyl ether precipitation (<20%). Accordingly, the fraction of the polymer under a critical molecular mass was unable to precipitate in the solvent. The average yield of the process was raised by purifying the polymer directly with dialysis using with membrane tubings (MWCO = 1 kDa) that were compatible with DMF solutions. In this way, PNVCL-COOH polymers were obtained without diethyl ether precipitation. The main drawback of this purification method is the inability to remove eventual traces of DMF that remain in water after dialysis. In a few polymers, it was possible to identify a minor peak at 2.9 ppm (Figure 2) that demonstrates the presence of a residual DMF in freeze-dried products. The peak had been previously mistaken for the methylene group (-C-S-CH2) present in the terminal group [35]. This hypothesis was excluded with the utilization of HSQC-DEPT heterocorrelated spectra (see Supplementary Figure S4). All polymers exhibited different LCSTs in relation to their different molecular mass and their corresponding M/I ratio.

**Figure 2.** HSQC spectra of PNVCL\_305.

#### *3.1. Structural Characterization of PNVCL-COOH Polymers*

NMR characterization was performed in D2O and d6-DMSO. In the 1H spectrum of PNVCL-COOH, four main signals were observed. The formation of the polymer was confirmed by the presence of broad signals and from the disappearance of the vinylic signals at 7.36 ppm. Similarly, amide I vibrations at 1631 and 1480 cm−1 (C-N stretching vibration) were observed, while the characteristic signals related to the monomer (C=C, 1658 cm−1 , CH= and CH2=, 3000 and 3100 cm−1 ) disappeared (see Supplementary Figure S5). All 1H NMR PNVCL-COOH spectra exhibited peaks at 1.77 ppm (3H, -CH2), 2.49 ppm (2H, -COCH2), 3.31 (2H, -NCH2) and 4.36 (1H, -NCH). The HSQC correlation spectrum with 1H and 13C assignments is reported in Figure 2. 1H and 13C spectra are provided in the Supplementary Materials (Figures S1 and S3). The presence of carboxyl end groups was confirmed by analyzing PNVCL\_244 and PNVCL\_305 in d6-DMSO using a high number of acquisitions. The signals were identified in small peaks observed at about 12 ppm in both spectra (Figure S2). In FT-IR spectra, the carboxyl end groups were recognized from the presence of broad signals at 3450 cm−1 (Figure S5). The carboxyl group content was estimated via conductimetric titration and was found to be inversely dependent on the M/I ratio. The number of terminations for PNVCL\_122, PNVCL\_305, PNVCL\_610, PNVCL\_1220 and PNVCL 1690 were, respectively, 0.96 ± 0.25, 0.73 ± 0.17, 0.63 ± 0.14, 0.55 ± 0.12 and 0.45 ± 0.13 mmol/g. 13C spectra confirmed the structure of the polymers based on the previous literature [10,12] (Figure 3 and Figure S2). Heterocorrelated 2D-HSQC spectra (see Supplementary Figure S4) allowed the identification of two minor signals at 1.55/29 ppm and 2.4/38 ppm that were related to the presence of the sulphur-bonded aliphatic methylene and the methylene bonded to the carboxyl termination group. The comparison between 13C spectra (Figure 3) of PNVCL\_122, PNVCL\_244 and PNCVL\_1220 in the range between 50 and 25 ppm confirmed that the signals are associated to the aliphatic portion of the termination groups. As shown, the signal intensity decreases as M/I ratio increases. This observation is in accordance with the presence of different PNVCL-COOH polymer with increasing molecular mass and increasing M/I ratio. The signals at 2.9/37 ppm were associated to CH3 residues of residual DMF solvent.

**Figure 3.** Comparison between 13C spectra of PNVCL\_122 (green), PNVCL\_305 (red) and PN-VCL\_1220 (blue).

#### *3.2. Spectroscopic Determination of LCST*

The miscibility curves were represented by plotting the transmittance of the solutions as a function of temperature (Figure 4a). LCSTs were determined from the inflection points of the sigmoidal functions that were used to fit the miscibility curves. PNVCL-COOH polymers (0.5 wt.% solutions) exhibited LCST in a range between 33 and 42 ◦C. The results showed that LCST diminishes in relation to the M/I ratios that were used for the synthesis of the polymers (Table 1). This molecular mass-dependent behavior is in accordance with a "classical" type I miscibility behavior. Accordingly, the increase of the hydrophobicity of the polymer results in the reduction of the LCST. The results excluded the presence of "step" transitions, that are generally observed in highly polydisperse polymers with different LCSTs. As a result, the variation of the M/I ratio provided a reliable procedure for the control of the LCST with simple FRP.

The type I behavior of PNVCL-COOH was further validated by measuring the LCSTs in the presence of salt species. The 0.5 wt.% PNVCL-COOH solutions were prepared in citrate (pH 3), acetate (pH 5) and phosphate buffers (pH 7). All buffers were prepared at the concentration of 0.1 M to compare the effect of the different ions on the LCSTs of the polymers. The results (Figure 4c) showed a strong dependence towards the types of ions dissolved in solution, as it was previously demonstrated by other studies on PNVCL polymers [4]. The results suggested that the ionic environment has a stronger effect in relation to the pH of the solution. The effect of salts was more pronounced for short chain polymers, which have a higher LCST (Figure 4c). The most evident effect was observed by dissolving PNVCL-COOH in phosphate buffer. This could be relevant from the biological point of view, since phosphate buffers solutions (e.g., PBS) are widely used in cell treatment kits [4,28–31]. During LCST transition, PNVCL-COOH undergoes a coil-to-globule transition [3,4]. Consequently, the lowering of the LCST due to the cell culture medium could result in potential cytotoxic effect due to the conformational change of the polymer. The effect of polymer concentration was assessed by measuring the LCSTs of PNVCL-COOH solutions at the concentration of 0.5, 0.1, 0.05 wt.% (Figure 4b). Normally, the LCST is associated with the point where the transmittance goes to zero [8,35,43]. However, this model was not suitable for describing the behavior of solutions at lower concentration (<0.1 wt.%). The determination of the LCST of dilute solution (<0.1 wt.%) of PNVCL has been previously reported with DLS, static light scattering and differential scanning calorimetry [4,44]. The calculation of the LCST at lower concentration was facilitated using a sigmoidal model for the interpretation of curves (Equation (1)). Dilution resulted in higher values of LCST and slower transition. Accordingly, PNVCL-COOH concentration

affects the kinetics and the energy of LCST transition. Finally, the LCSTs were measured in presence of NaCl in range of concentrations between 0 and 0.15 M (Figure 4d). Results showed that LCST decreases proportionally in relation to NaCl concentration [45]. LCST decreased by about 1 ◦C in a physiological solution (0.9% NaCl, or 0.15 M). Consequently, pharmaceutical applications of PNVCL-COOH polymer in a physiological solution would require a polymer with a LCST of at least > 38 ◦C in order to avoid cytotoxic effects due to the polymer transition.

**Figure 4.** (**a**) Miscibility curves (transmittance vs. temperature) of PNVCL-COOH polymers at the concentration of 0.5 wt.% in milliQ water (pH = 6.20). (**b**) Variation of LCST reported as a function of the PNVCL-COOH concentration in milliQ water (pH = 6.20). (**c**) Variation of LCST of 0.5 wt.% PNVCL-COOH solutions associated to different type of buffers. (**d**) Variation of LCST of 0.5 wt.% PNVCL-COOH solutions (milliQ water, pH = 6.20) associated to the concentration of NaCl.

#### *3.3. Scattering Determination of LCST*

DLS provided lower values of LCST in relation to UV-VIS spectroscopy. The analyses of polymers with higher M/I (PNVCL\_610, PNVCL\_1220, PNVCL\_1690) provided a direct measurement of LCST from the displacement of the autocorrelation curve due to the transition. The analyses of the other samples did not provide a direct estimation due to the small dimensions of the PNVCL-COOH macromolecules in solution. Consequently, the LCST was estimated by checking visible turbidity. The observed differences between DLS and UV-VIS measurement were related to the polydispersity of the polymers. DLS is more sensitive to the formation of aggregates in the proximity of LCST. The formation of globular aggregates at temperature inferior to LCST is related to the fraction of PNVCL-COOH polymers with higher molecular mass. Since the scattering intensity is proportional to Dh 6 , a small fraction of polymer undergoing coil-to-globule transition is able to produce variation in the autocorrelation curve shape or position (Figure S7) [39]. Accordingly, the entity of the differences between UV-VIS and DLS measurements provided an overview on the polydispersity of PNVCL-COOH polymers. According to the results, the polymer with highest polydispersity was PNVCL\_610.

#### *3.4. Determination of Molecular Mass*

The molecular weight determination of polymer was carried out by SEC, DLS and viscosimetry. DLS and viscosimetry provided an estimation of the average molecular mass, while SEC did not produce any appreciable results due to the sorption of PNVCL on the column. This issue has been previously addressed for the analysis of PNVCL column in most solvents, including aqueous buffers and THF [10,46]. DLS allowed the estimation of the molecular mass according to the models described by Lau [40] and Eisele [41]. The viscosimetric molecular mass of PNVCL\_244, PNVCL\_305 and PNVCL\_1220 mass was determined using water as a solvent at 25 ◦C. Samples were analyzed in dilute conditions (≤ 0.2 wt.%) in order to prevent the formation of foam. Due to the low viscosity of the sample, the differences between the run times were very small. Similarly, the values of inherent viscosity (*ηinh*) were considered too low for the calculation of the molecular mass with the model described by Kraemer [47]. The comparison between DLS and viscosimetric measurements is reported in Table 1. The estimated molecular mass as a function of the M/I value is reported in Figure 5a and the variation of LCST as a function of the molecular mass is reported in Figure 5b. Both DLS and viscosimetric results are in line with a type I thermoresponsive polymer behavior. As the molecular mass increases, the LCST decreases. The utilization of K and a constant reported by Kirsh [12,42] demonstrated that the two models can be equally applied for PNVCL-COOH polymers in this molecular weight range (15–50 kDa). Molecular mass values obtained through viscosimetry were slightly lower than those obtained by interpreting DLS data with the model provided by Lau (Equation (2)), while the use of Eisele's equation led to the overestimation of the molecular mass. Interestingly, it was observed that the values calculated using Lau's equation were almost half the values obtained with Eisele's. The bigger differences between DLS and viscosimetry data are observed with increasing molecular mass (Table 1, Figure 5a,b). Accordingly, scattering methods should be considered for the estimation of the molecular mass of PNVCL polymers with lower molecular mass, that are not suitable for viscosimetric analysis due to their low viscosity. The reported relations between LCST and *Mη* are in accordance with the mixing behavior of aqueous PNVCL solutions reported by Meeussen [22] and Kirsh [42]. Accordingly, the small difference between the calculated LCST may be related to the different end groups of the polymers under consideration. The presence of terminations or compounds that increase the hydrophilicity of PNVCL is known to increase the LCST [27,48]. Furthermore, PNVCL-COOH polymers with a 32◦C LCST have been frequently used as starting polymers for the preparation of thermoresponsive particles [7–9,19,35,38]. This LCST value has been previously associated to the behavior of PNVCL-COOH polymers with a molecular weight of 1 kDa by means of GPC-SEC measurements in THF [35] and never discussed in subsequent publications [7–9,16–19].

PNVCL\_1690 shows similar properties as the LCST is close to 33 ◦C. According to DLS measurements, PNVCL-COOH polymers require a molecular mass higher than 100 kDa in order to exhibit LCST at 32 ◦C. Similarly, viscosimetric analyses demonstrated that the value should be at least higher than 42 kDa, which is the value that we associated to a LCST of 34 ◦C. Accordingly, the importance of the molecular mass of PNVCL polymers appeared to be underestimated in studies oriented towards biological applications [7–9,16–19,35]. The molecular mass of the PNVCL has a central role in defining the thermoresponsive behavior of nanoparticles, microgels, gels and other biocompatible devices for drug and gene delivery. Consequently, it is essential to correctly characterize the molecular mass with precise, standardized, and reproducible methods.

**Figure 5.** (**a**) Average molecular weight as a function of M/I ratio. (**b**) Spectroscopic LCST reported as a function of the average molecular weight. The graphics show the comparison between the values calculated using the models reported by Lau (black), Eisele (blue), and viscosimetric masses calculated using Equations (6) (red) and (7) (violet).

#### *3.5. Determination of LCST in Human Plasma*

The critical miscibility behavior of PNVCL-COOH was observed in human plasma to evaluate possible cytotoxic effects related to the utilization of the polymer in physiological fluids. Plasma represents 55% of the blood and consists mainly of water, dissolved ions, proteins and gases. Among the main proteins, albumin, fibrinogen and globulins are present, whose functional groups give rise to the intense visible bands in the region between 200 and 600 nm [49]. In both samples analyzed, the peak at 576 nm and the shoulder at 540 nm are related to the presence of oxyhemoglobin [50] due to the hemolysis of residual erythrocytes. A first calibration of the matrix was performed on a pool of plasma realized from the union of samples taken from 12 heathy donors in a temperature range between 25 and 50 ◦C. The calibration excluded any spectral modification between 25 and 42 ◦C. Due to the complexity of the matrix, all solutions were analyzed using milliQ water as a reference. The measurements of the LCST of PNVCL\_122, PNVCL\_244 and PNVCL\_305 were provided by the analysis of the region devoid of amino acid signals, between 600 and 800 nm (Figure 6a and Figures S8,S9). Upon heating, the absorbance signal of the spectra was shifted towards higher values due to the conformational change of the polymers. The results show that the human plasma is responsible for a significant lowering of the LCST by about 10 ◦C, as spectral changes were already observed at 28 ◦C. This is in line with the results of previous experiments, which have shown that LCST is affected by the presence of ions and proteins. By heating up the solution to 37 ◦C, the spectra change dramatically, and it was no longer possible to recognize any spectral information. However, the spectral properties of plasma were restored by cooling the system back to 25 ◦C. Accordingly, the reversibility of PNVCL-COOH transition is maintained within the plasma matrix. LCST values were calculated by fitting the transmittance vs. temperature data with a sigmoidal function (Equation (1)). Transmittance data and fitting functions are reported in Figure 6b. The comparison between the LCST of PNVCL-COOH in milliQ water and plasma are reported in Table 2. It can be concluded that the polymer is not suitable for direct utilization within the plasma at the concentration of 0.5 wt.%. However, the reversibility of the transition could be an important starting point for future developments. According to the previously reported results, the LCST can be increased by simply decreasing the chain length by modifying the M/I ratio and by diminishing polymer concentration.

**Figure 6.** (**a**) Variation of UV-VIS spectrum of a solution of PNVCL\_244 in human plasma (0.5 wt.%) with temperature. (**b**) Transmittance of PNVCL\_122 (black squares), PNVCL\_244 (blue circles) and PNVCL\_305 (red triangles) solution in human plasma (0.5 wt.%) calculated at 800 nm reported as a function of temperature. The graph also shows the sigmoidal fitting functions that were used for the calculations of the LCST.

**Table 2.** Comparison between the LCST of PNVCL-COOH polymers (0.5 wt. %) in milliQ water and in human plasma.

#### **4. Conclusions**

In this work, the preparation of PNVCL-COOH linear thermoresponsive polymers with a LCST of between 33 and 42 ◦C was reported. The results contradict the established thesis that PNVCL has a characteristic LCST at 32 ◦C and exhibits a miscibility behavior similar to PNIPAM. The utilization of different ratios between monomer and initiator (M/I) allowed to lower the LCST by increasing the molecular mass of the polymer. Accordingly, the increase in molecular mass was associated with a decrease in terminal groups content that was observed in NMR spectroscopy and conductivity titration. Molecular mass characterization was approached with different method and a comparison of the results was provided. While GPC-SEC has proven to be unreliable regarding the tendency of the polymer to adsorb on the column, DLS and viscosimetry proved to be simple and effective methods for the estimation of the molecular mass in simple aqueous solution. The behavior of the polymer was described according to its concentration and in the presence of different environments, such as buffers, NaCl solutions and human plasma. The utilization of a sigmoidal model allowed to correlate the equilibrium miscibility temperature (LCST) as the inflection point of the miscibility curve. This allowed to interpret with greater precision the behavior in diluted solutions or in complex matrices. The variability associated with the LCST values in the different solutions demonstrated the importance in reporting the LCST

of PNVCL polymers in relation to their concentration and molecular mass. In addition, the study demonstrates the importance of the screening of the behavior of PNVCL polymers in biologically relevant environments (plasma, PBS, physiological solution). The LCST of PNVCL polymers for biological applications should be determined within these solutions in order to prevent cytotoxic effects due to thermo-induced conformational change.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/polym13162639/s1, Figure S1: 1H spectrum of PNVCL\_122; Figure S2: Carboxylic termination group signals in 1H NMR spectrum of PNVCL\_305 dissolved in d6 -DMSO; Figure S3: 13C spectrum of PNVCL\_122; Figure S4: HSQC-DEPT spectrum of PNVCL\_122; Figure S5: Comparison between NVCL and PNVCL-COOH spectrum (PNVCL\_305); Figure S6 Reduced viscosity reported as a function of PNVCL\_244 (black squares), PNVCL\_305 (red circles) and PNVCL\_1220 (blue triangles) concentration. The value of *ηred* were calculated according to the Huggins method by using the calibration lines reported in the figure; Figure S7: Displacement of the correlation curve of PN-VCL\_1220 solution (0.5 wt.%) associated to LCST transition; Figure S8: Variation of UV-VIS spectrum of a solution of PNVCL\_122 in human plasma (0.5 wt.%) with temperature; Figure S9: Variation of UV-VIS spectrum of a solution of PNVCL\_305 in human plasma (0.5 wt.%) with temperature; Figure S10: Conductimetric titration of PNVCL\_305.

**Author Contributions:** Funding acquisitions: G.T.; investigation: L.M.; methodology: L.M., F.B., G.E. and I.D.; supervision: G.T.; validation: M.D.B. and F.B; writing—original draft: L.M.; writing review and editing: M.D.B. and F.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Italian Ministry of Health (Ricerca Corrente).

**Institutional Review Board Statement:** Not Applicable.

**Informed Consent Statement:** Not Applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors are grateful to Paolo Macor for the help and the cooperation and to Josie McQuillan for her valuable assistance in revising English language.

**Conflicts of Interest:** The authors declare no conflict of interest in this publication.

#### **Abbreviations**

#### **References**

## *Article* **Temperature/Reduction Dual Response Nanogel Is Formed by In Situ Stereocomplexation of Poly (Lactic Acid)**

**Wenli Gao † , Zhidan Wang † , Fei Song, Yu Fu, Qingrong Wu and Shouxin Liu \***

> Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710119, China<EMAIL_ADDRESS>(W.G<EMAIL_ADDRESS>(Z.W<EMAIL_ADDRESS>(F.S<EMAIL_ADDRESS>(Y.F<EMAIL_ADDRESS>(Q.W.)

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +86-29-81530781

† These authors contributed equally to this work.

**Abstract:** A novel type of dual responsive nanogels was synthesized by physical crosslinking of polylactic acid stereocomplexation: temperature and reduction dual stimulation responsive gels were formed in situ by mixing equal amounts of PLA (Poly (Lactic Acid)) enantiomeric graft copolymer micellar solution; the properties of double stimulation response make it more targeted in the field of drug release. The structural composition of the gels was studied by proton nuclear magnetic resonance ( 1H NMR) and Fourier transform infrared spectroscopy (FT-IR). Using transmission electron microscope (TEM) and dynamic light scattering (DLS) instruments, the differences in morphology and particle size were analyzed (indicating that nanogels have dual stimulus responses of temperature sensitivity and reduction). The Wide-Angle X-ray diffractionr (WAXD) was used to prove the stereocomplexation of PLA in the gels, the mechanical properties and gelation process of the gels were studied by rheology test. The physically cross-linked gel network generated by the self-recombination of micelles and then stereo-complexation has a more stable structure. The results show that the micelle properties, swelling properties and rheological properties of nanogels can be changed by adjusting the degree of polymerization of polylactic acid. In addition, it provides a safe and practical new method for preparing stable temperature/reduction response physical cross-linked gel.

**Keywords:** stereocomplexation; polylactic acid; temperature/reduction; self-recombination

#### **1. Introduction**

In the field of drug delivery, there are many types of polymer-based carriers, such as nanoparticles [1], vesicles [2,3], polymer micelles [4–7], hydrogels [8–10], etc. Among these carriers, polymeric micelles are one of the most common. However, the instability of the micelle structure causes it to dissociate at a concentration lower than the critical micelle concentration, which will lead to the loss of drugs during the blood circulation and limit its further clinical application [11,12]. Scientists prepared polymers for improving the physical stability of polymer micelles through various cross-linking methods. Nanohydrogel is one of them. As one of the classifications of hydrogels, nanohydrogels are defined as a nano-scale polymer with 3D network nanoparticles, this network is formed by crosslinking polymer chains swelled in a good solvent [13,14]. Compared with commonly used nanocarriers (such as micelles), nanohydrogels contain a large amount of water in a swollen state, and stably load drugs, reducing the loss of drugs during blood circulation.

Generally speaking, hydrogels can be divided into two types according to crosslinking methods, one is a chemical crosslinking hydrogel [15], the other is a physical crosslinking hydrogel [16]. The chemical crosslinking hydrogel is usually crosslinked by chemical crosslinking agent and the force of cross-linking is the covalent bond generated by the functional groups between the polymer chains, so it has better mechanical

**Citation:** Gao, W.; Wang, Z.; Song, F.; Fu, Y.; Wu, Q.; Liu, S. Temperature/Reduction Dual Response Nanogel Is Formed by In Situ Stereocomplexation of Poly (Lactic Acid). *Polymers* **2021**, *13*, 3492. https://doi.org/10.3390/ polym13203492

Academic Editor: Mattia Sponchioni

Received: 30 August 2021 Accepted: 7 October 2021 Published: 12 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

strength and stability than physically cross-linked hydrogels [17,18]. However, the addition of a chemical crosslinking agent may lead to biodegradable hydrogels become a huge problem [19]. Physical cross-linking hydrogels mainly use intermolecular hydrogen bonding as the main cross-linking force, avoiding the formation of undegradable bonds, and has better biocompatibility, degradability and application prospect than chemical cross-linked hydrogels. The polylactic acid stereocomplex has many advantages. Due to its extraordinary biological, thermal, and mechanical properties, it shows great potential as a biological material. The group of Sytze J. Buwalda conducted research to prepare PEG-PLA star-shaped block copolymer hydrogels through physical crosslinking combined with photopolymerization. The gel is degraded by the hydrolysis of the ester groups in the PLA chain, resulting in the loss of physical and chemical crosslinks. This study shows that the injectable, photocrosslinked PEG-PLA star block copolymer hydrogel is a promising material for biomedical applications such as long-term controlled drug delivery [20].

The broad application prospects of stimulus responsive hydrogels have led to various single-response [21], double-response and multi-response hydrogels being studied and applied [22–24]. However, there are few reports on reduction/temperature dual response hydrogels. In recent years, biodegradable hydrogels with degradable bonds have become a hot topic [25,26]. Scientists have introduced degradable bonds such as hydrazine [27], enzymes [28,29], disulfide bonds [30], etc. into polymer molecules to achieve the synthesis of degradable hydrogels. Among them, disulfide bonds are favored by scientists because they can be cleaved and reformed under relatively simple and mild conditions [31,32]. In this article, we first synthesize an initiator 2-((2-Hydroxyethyl) disulfanyl) ethyl methacrylate (HSEMA) with disulfide bond through the monoesterification reaction of 2, 2′ -dithiodiethanol and methacryloyl chloride, and then used it for ring-opening polymerization of poly(lactide) to form macromonomer HSEMA-PLLA and HSEMA-PDLA, the temperature-sensitive monomers 2-(2-methoxyethoxy) ethyl methacrylate (MEO2MA) and oligo (ethylene glycol) methacrylate (OEGMA) undergo free radical polymerization with macromonomer HSEMA-PLLA or HSEMA-PDLA under the condition of AIBN as the initiator to form temperature/reduction dual responsive micelles. Mixing equal amounts of PLA enantiomeric micellar solution in situ generates a temperature reduction dual stimulus responsive gel. The physically cross-linked gel network generated by the self-recombination of micelles and then stereo-complexation provides a safe and practical new method for the preparation of temperature/reduction dual responsive physical cross-linking gel.

#### **2. Materials and Methods**

#### *2.1. Materials*

L-lactide, D-lactide (99.0%) and methacryloyl chloride (95%, *M*n = 104.53 g·mol−1 ) were purchased from Macleans (Shanghai, China). Oligo (ethylene oxide) methacrylate (OEGMA, 95%, *M*n = 475 g·mol−1 ) and 2-(2-methoxy ethoxy) ethyl methacrylate (MEO2MA, 95%, *M*n = 188.22 g·mol−1 ) were obtained from TCI (Shanghai Development Co., Ltd., Shanghai, China). 2,2′ -dithiodiethanol (90%, *M*n = 154.25 g·mol−1 ) was purchased from Alfa Aesar (Shanghai, China). 1,4-dithiothreitol (DTT, 99.0%, *M*n = 154.25 g·mol−1 ) was purchased from Rhawn (Shanghai, China), Tin(II) 2-ethylhexanoate (99%, *M*n = 405.12 g·mol−1 )was purchased from J&k (Beijing, China), 2,2′ -Azoisobutyronitrile(AIBN) ( Macleans, Shanghai, China) was purified by recrystallization from methanol. The solvents used are all dried and double distilled water is used in aqueous solutions.

#### *2.2. Synthesis of 2-((2-Hydroxyethyl) Disulfanyl) Ethyl Methacrylate (HSEMA)*

2-((2-Hydroxyethyl) disulfanyl) ethyl methacrylate (HSEMA) was synthesized by the monoesterification reaction of 2,2′ -dithiodiethanol and methacryloylchloride [33,34]. The synthesis route was as follows: 2,2′ -dithiodiethanol (4 mmol, 0.542 mL) and triethylamine (1 mL) were completely dissolved in 20 mL dried THF. Then methacryloyl chloride (4 mmol, 0.407 mL) was added drop-wise for 20 min in an ice bath at 0 ◦C, the reactants were stirred at room temperature for 24 h. The produced white solids, i.e., triethylamine

salt, were removed using the method of filtration. Subsequently, the solvent was removed by rotary evaporation, and the product was purified by column chromatography.

′

′

#### *2.3. Synthesis of Macromonomer HSEMA-PDLA*

Synthesis of macromonomer HSEMA-PLLA/HSEMA-PDLA used the method of ringopening polymerization with HSEMA as the initiator and lactide as the raw material. The synthesis route was as follows: D-lactide (6.9 mmol, 1 g), initiator HSEMA (0.53 mmol) and dry toluene (15 mL) were added to a dry and clean three-necked flask (Scheme 1). After the mixture were fully dissolved, the catalyst Tin (II) 2-ethylhexanoate (Sn(Oct)2) (55 µL) was added. The reaction system was refluxed at 120 ◦C for 24 h under a nitrogen atmosphere, and then the crude products were precipitated and purified twice in cold anhydrous ether. Finally, the products were stored in a vacuum drying cabinet at room temperature. The synthesis of HSEMA-PLLA was similar to HSEMA-PDLA. μ

**Scheme 1.** Initiator HSEMA (**a**), macromonomer HSEMA-PLLA/PDLA (**b**), graft copolymer P(MEO2MA-*co*-OEGMA)-*g*-(HSEMA-PDLA) (**c**), Schematic diagram of the synthesis and hydrogel complex mechanism (**d**).

#### *2.4. Synthesis of Graft Copolymer P(MEO2MA-co-OEGMA)-g-(HSEMA-PLLA)*

The graft copolymer P (MEO2MA-co-OEGMA)-g-(HSEMA-PLLA) was synthesized by free radical polymerization with 2-(2-methoxy ethoxy) ethyl methacrylate (MEO2MA) and oligo (ethylene oxide) methacrylate (OEGMA) as temperature-sensitive monomers, HSEMA-PLLA as macromonomers and 2,2′ -Azoisobutyronitrile (AIBN) as the initiator. The synthesis route was as follows: the macromonomer HSEMA-PLLA and DMF (3 mL) were added to a dry and clean Shrek tube. After HSEMA-PLLA were fully dissolved, MEO2MA, OEGMA and initiator AIBN were added to the mixed solution. The reaction

system was reacted at 70 ◦C for 6 h under nitrogen atmosphere. The crude products were precipitated and purified twice in ice anhydrous ether, then dialyzed at room temperature for 3 days. Finally, the products were freeze-dried and stored in a vacuum drying cabinet at room temperature.

#### *2.5. Synthesis of Temperature/Reduction Nanogels*

Copolymers of equal mass were respectively dissolved in water to obtain a uniform single enantiomeric graft copolymer solution. Then the enantiomeric graft copolymer solutions were mixed in equal volumes at 37 ◦C to obtain a stereocomplexed gel [35,36]. The complex mechanism was shown in Scheme 1.

#### *2.6. Characterization of the Samples*

#### 2.6.1. Nuclear Magnetic Resonance Spectroscopy (1H-NMR)

The structure of the compound was characterized using a Bruker-300MHz spectrometer (300 MHz Avance, Bruker Corporation, Karlsruhe, Germany) with CDCl3 as a solvent and tetramethylsilane (TMS) as an internal standard at room temperature.

#### 2.6.2. Fourier Transform Infrared Spectroscopy (FT-IR)

The functional group of the compound was characterized by Fourier transform infrared spectroscopy (FTIR) (Tensor 27, Bruker Corporation, Karlsruhe, Germany). Before the measurement, the sample was uniformly mixed with dry KBr, dried in a vacuum drying oven for 24 h, and finally pressed at room temperature.

#### 2.6.3. The Wide-Angle X-ray Diffraction (WAXD)

The wide-angle X-ray diffraction (WAXD) analysis was performed on Ni-filtered Cu Kα (λ = 0.154 nm) at 25 ◦C on the D8 Advance diffractometer from Bruker, Karlsruhe, Germany, with a 2*θ* scanning rate of 2◦/min and a scanning range of 5–40◦ .

#### 2.6.4. Low Critical Solution Temperature (LCST)

The LCST of the gels were measured by UV-V is spectroscopy (TU-1901, Beijing Purkinje General Instrument Corporation, Beijing, China). A series of nanogel solutions (2.00 mg/mL) was arranged in a transparent sample vial. The transmittance of the nanogels was measured using the TU-1901 at a wavelength of 660 nm, photometric mode, and a temperature range of 20–50 ◦C.

#### 2.6.5. The Scanning Electron Microscope (SEM) Analysis

The gels samples were immersed in the distilled water at 25 ◦C till swell-equilibrium. The swollen gels were quickly frozen in liquid nitrogen and then freeze-dried in a freeze dryer. Microscopic morphology of the gel was performed on a desktop scanning electron microscope (SEM, TM 3030 Hitachi, Tokyo, Japan) in low vacuum mode. Before the observation, in order to improve the conductivity of the sample, it was subjected to gold spray treatment for 80 s.

#### 2.6.6. Dynamic Light Scattering (DLS) Analysis

Laser particle size meter (BI-90Plus, Brookhaven, New York, United States) was used to measure the particle size of nanogels under different conditions: 1 mg/mL of solution was prepared and filtered by 0.45 µm water filter. The particle size was measured at room temperature and variable temperature, respectively.

#### 2.6.7. Swelling Kinetics of the Gel

The preparation method of the dried gel used for the swelling performance test is: 0.5 g enantiomeric copolymer was put in a sample bottle respectively, add THF right amount to dissolve completely. In an ice bath, the THF solution of the copolymer was dripped drop by drop into a round-bottomed flask containing 5 mL of secondary water. Ultrasound at 4 ◦C until the solution reaches equilibrium (about 30 min), distilled THF in the solution under reduced pressure at room temperature to obtain a uniform enantiomeric graft copolymer micelle solution with a mass fraction of 10 *w*/*v*%, after mixing the same amount of enantiomeric copolymer solution and stirring for 40 min, ultrasonic for 30 min to complete the complexation, heat up in the temperature range of 25–70 ◦C and volatilize the solvent and obtain a completely dry PLA-based gels.

The swelling kinetics and de-swelling kinetics of the gel were explained by the weighing method. The specific method of the swelling kinetics is as follows: fully dried gels were soaked in the secondary water at 25 ◦C, taken out, the mass was weighed three times after a certain period of time, and the water on the surface of the gel was absorbed by filter paper. Taking the average of the data, its swelling ratio was calculated according to the following formula.

$$\left[\text{Swelling ratio}\right] = \left(\mathbf{W\_t} - \mathbf{W\_d}\right) / \mathbf{W\_d} \tag{1}$$

where Wd is the mass of dry gel (g), Wt is the mass of the gel taken out at a fixed time point during the swelling process (g).

#### 2.6.8. Transmission Electron Microscopy (TEM) Analysis