*Article* **Synthesis and Characterization of Hybrid Materials Derived from Conjugated Copolymers and Reduced Graphene Oxide**

**Alexandros Ch. Lazanas <sup>1</sup> , Athanasios Katsouras <sup>1</sup> , Michael Spanos 1,2, Gkreti-Maria Manesi <sup>1</sup> , Ioannis Moutsios 1,3 , Dmitry V. Vashurkin 4,5, Dimitrios Moschovas <sup>1</sup> , Christina Gioti <sup>1</sup> , Michail A. Karakassides <sup>1</sup> , Vasilis G. Gregoriou <sup>2</sup> , Dimitri A. Ivanov 3,4,5 , Christos L. Chochos 1,2,\* and Apostolos Avgeropoulos 1,4,\***

	- 68057 Mulhouse, France <sup>4</sup> Faculty of Chemistry, Lomonosov Moscow State University, 9MSU, GSP-1, 1-3 Leninskiye Gory, 119991 Moscow, Russia
	- 5 Institute of Problems of Chemical Physics, Russian Academy of Sciences, Chernogolovka, 142432 Moscow, Russia
	- **\*** Correspondence: chochos@eie.gr (C.L.C.); aavger@uoi.gr (A.A.); Tel.: +30-26-5100-9001 (A.A.)

**Abstract:** In this study the preparation of hybrid materials based on reduced graphene oxide (rGO) and conjugated copolymers is reported. By tuning the number and arrangement of thiophenes in the main chain (indacenothiophene or indacenothienothiophene) and the nature of the polymer acceptor (difluoro benzothiadiazole or diketopyrrolopyrrole) semiconducting copolymers were synthesized through Stille aromatic coupling and characterized to determine their molecular characteristics. The graphene oxide was synthesized using the Staudenmaier method and was further modified to reduced graphene oxide prior to structural characterization. Various mixtures with different rGO quantities and conjugated copolymers were prepared to determine the optoelectronic, thermal and morphological properties. An increase in the maximum absorbance ranging from 3 to 6 nm for all hybrid materials irrespective of the rGO concentration, when compared to the pristine conjugated copolymers, was estimated through the UV-Vis spectroscopy indicating a differentiation on the optical properties. Through voltammetric experiments the oxidation and reduction potentials were determined and the calculated HOMO and LUMO levels revealed a decrease on the electrochemical energy gap for low rGO concentrations. The study indicates the potential of the hybrid materials consisting of graphene oxide and high band gap conjugated copolymers for applications related to organic solar cells.

**Keywords:** hybrid materials; conjugated copolymers; rGO; HOMO; LUMO; indacenothiophene; indacenothienothiophene; cyclic voltammetry

### **1. Introduction**

Over the last two decades graphene [1] has made an impressive impact on current technology due to its remarkable properties. The oxidized form of graphene, namely graphene oxide (GO), has been known for almost two centuries [2], and despite its impressive features, it lacks the electronic properties of graphene due to the mixed sp<sup>3</sup> and sp<sup>2</sup> orbital attributed to the oxygen groups. Many efforts have been conducted to reduce the oxygen groups using reductive substances such as NaBH<sup>4</sup> etc. [3,4]. This allotropic form of carbon is generally known as reduced graphene oxide (rGO) and constitutes a fairly good compromise between the outstanding electronic properties of graphene and the excellent solubility of graphene oxide. This fact is allocated to the reduction process which is not exclusively completed (not 100% yield of the process) and the produced rGO

**Citation:** Lazanas, A.C.; Katsouras, A.; Spanos, M.; Manesi, G.-M.; Moutsios, I.; Vashurkin, D.V.; Moschovas, D.; Gioti, C.; Karakassides, M.A.; Gregoriou, V.G.; et al. Synthesis and Characterization of Hybrid Materials Derived from Conjugated Copolymers and Reduced Graphene Oxide. *Polymers* **2022**, *14*, 5292. https://doi.org/ 10.3390/polym14235292

Academic Editor: Youngmin Lee

Received: 8 November 2022 Accepted: 29 November 2022 Published: 3 December 2022

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

**Copyright:** © 2022 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/).

still retains some of the aforementioned oxygen groups that enable stable dispersions with hydrophobic interactions, since rGO is not water-soluble similar to its precursor [5].

The above-mentioned characteristics strongly suggest that rGO could readily form films for a great number of applications such as organic photovoltaics [6,7], sensors [8] etc. To that end, many groups have endeavored the combination of conjugated polymers and rGO [9–11] to address the fact that their possible π-π stacking with graphene and rGO enables the fine-tuning of the optoelectronic polymers. Such an approach leads to the opportunity for a flexible and adjustable system which may solve the relatively low photovoltaic efficiencies that these polymers commonly exhibit [12,13]. As a result, a wide range of applications based on composite materials consisting of graphene oxide in polymer matrices have been reported in the literature. These systems have already been used as membranes [14,15], energy devices [16], supercapacitors [17], drug delivery [18] flexible and/or wearable electronics [19] etc.

The non-covalent attachment between the conjugated polymer and the rGO has significant advantages. The rGO is added to the polymer solution and the mixture is agitated through sonication. This approach leads to the stabilization through non-covalent interactions which do not induce intrinsic changes in the chemical structures, electronic or mechanical properties of the rGO. The π-π interactions between the rGO and the conjugated backbone render the hybrid materials capable of being easily processed due to the rGO solubility. The exquisite properties presented on the specific hybrid copolymers have paved the way for their extensive use in photovoltaic devices using facile methods [20–23].

Herein, our focus was the synthesis of high band gap conjugated polymers [24] based on indacenothiophene and indacenodithienothiophene [25] through the Stille aromatic coupling method. These monomers play the role of donors, in *donor-acceptor* units with varying acceptor units. The characterization through Gel Permeation Chromatography (GPC) indicated the molecular features of the copolymers such as number average molecular weight and dispersity (Ð). In addition, we report the synthesis of graphene oxide from graphite powder using a modified Staudenmaier method [2] and its further reduction to obtain the reduced graphene oxide [3]. X-ray Diffraction Analysis (XRD) was carried out to assess the characteristic properties before and after the oxygen incorporation between the graphene oxide sheets. The preparation of mixtures consisting of conjugated polymers and reduced graphene oxide is expected to have a significant role in the fine tuning of their energy gaps and possibly revolutionize various electronic applications such as organic solar cells, OLED displays etc. The materials were mixed in an appropriate dispersing medium (ortho-dichlorobenzene or o-DCB) and sonicated for 6 h prior to their characterization. Specifically, the hybrid dispersions were morphologically characterized using Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS). Their optoelectronic properties were evaluated with UV-Vis Spectroscopy and Cyclic Voltammetry (CV). Also, the thermal stability of the involved materials (GO, rGO, conjugated copolymers, hybrid materials) was verified through thermogravimetric analysis (TGA).

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

#### *2.1. Materials*

All monomers (thiophene-based, difluorobenzothiadiazole and diketopyrrolopyrrole) and solvents (toluene, methanol, acetone, hexane, acetonitrile, ortho-dichlorobenzene) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. All reagents for the preparation of the samples [tris (dibenzylideneacetone)dipalladium (0) or Pd2dba3, tri (o-tolyl)phosphine or P (o-Tol)3, powdered graphite, sulfuric acid, nitric acid, potassium chlorate powder and sodium borohydride or NaBH4] were purchased from Fluka (Fluka Chemie GmbH, Buchs, Switzerland) and used without additional manipulations.

#### *2.2. Methods*

Number average molecular weights (*Mn*) and dispersity indices (Ð) were determined with Gel Permeation Chromatography (GPC) at 150 ◦C on a high temperature PL-GPC 220 system using a PL-GEL 10 µm guard column, three PL-GEL 10 µm Mixed-B columns and o-DCB as the eluent. The instrument (Agilent Technologies, St. Clara, CA, USA) was calibrated with narrow polystyrene standards (Mp ranged from 4830 g/mol to 3,242,000 g/mol).

UV/vis spectra were measured on a Cary 5000 UV-Vis-NIR (Agilent Technologies, St. Clara, CA, USA) in dual beam mode.

Raman spectroscopy studies were conducted through a micro-Raman RM 1000 Renishaw system (Renishaw, Wotton-under-Edge, UK). The power of the laser was 30 mW and a 2 µm focus spot was utilized.

X-ray diffraction (XRD) measurements were carried out in a Bruker D8 Advance (Bruker, Billerica, MA, USA) with Bragg–Brentano geometry with LYNXEYE detector in 2θ range from 2◦ to 30◦ . For the X-rays, a Cu-K<sup>α</sup> wire was used, resulting in a radiation of 1.5406 Å wavelength.

Thermogravimetric analysis (TGA) was carried out in a Perkin Elmer Pyris-Diamond instrument (PerkinElmer, Inc., Waltham, MA, USA). Approximately 10 mg of each sample (graphene oxide, reduced graphene oxide, pristine conjugated copolymers and final hybrid materials) was heated from 40 ◦C to 800 ◦C following a step of 5 ◦C/min under nitrogen atmosphere.

Scanning Electron Microscope (SEM) images were obtained on gold coated samples (Polaron SC7620 sputter coater, Thermo VG Scientific, Waltham, MA, USA) using a JEOL JSM6510LV scanning electron microscope equipped with an INCA PentaFETx3 (Oxford Instruments, Abingdon, UK) energy dispersive X-ray (EDX) spectroscopy detector. Data acquisition was performed with an accelerating voltage of 20 kV and 60 s accumulation time, respectively. The samples were prepared using an appropriate solvent for solution casting and then dried overnight in a vacuum oven. Prior to the morphological characterization a gold/palladium target was used to sputter the samples utilizing the SC7620 Mini Sputter Coater/Glow Discharge System (Quorum technologies, Sacramento, USA). Also, additional experiments were conducted on a specific copolymer (copolymer-C2 with 5 wt% and 10 wt% rGO) in order to study its morphological behavior in thin film state. The samples were prepared using spin coating onto silicon wafer under ambient conditions (spinning velocity ~3000 rpm for approximately 30 s, polymer solution in o-DCB concentration equal to 3%).

Voltammetric Experiments were performed with a PGSTAT12 electrochemical analyzer (Metrohm Autolab, Utrecht, Netherlands) in a single compartment three-electrode cell. Bare or modified GCEs (IJ Cambria, Swansea, UK) were used as working electrodes and a platinum wire served as the auxiliary electrode. The reference electrode was an Ag/AgCl 3M KCl (IJ Cambria, Swansea, UK) electrode and all potentials hereafter are quoted to the potential of this electrode. Cyclic Voltammograms (CVs) were recorded in 0.1 mol L−<sup>1</sup> Bu4NBF<sup>4</sup> in solvent mixture acetonitrile (ACN):o-DCB at a scan rate of 0.05 V s−<sup>1</sup> . All potentials are quoted after the Fc/Fc<sup>+</sup> redox couple as explained in the experimental section.

#### *2.3. Synthesis of Hybrid Materials*

Three (3) conjugated copolymers using thiophene-based and difluoro benzothiadiazole or diketopyrrolopyrrole as monomers were synthesized based on the Stille aromatic coupling in a process that has been already described by our group [26] in order to be used as matrices for the preparation of hybrid materials. In a three-neck round bottom flask, fitted with a condenser, 3 cycles of argon/vacuum were conducted. Afterwards, the donorand the acceptor-type monomers were added along with Pd2dba3, which is the catalyst (22.90 mg), and P (o-Tol)<sup>3</sup> as a ligand (60.87 mg) and 3 cycles of argon/vacuum were carried out again. Finally, toluene (20 mL) was added, and 3 cycles of argon/vacuum were again performed. The reaction mixture is allowed to react for at least 48 h at 120 ◦C.

Upon completion of the polymerization, the synthesized material was precipitated in 500 mL of methanol and filtered on a paper filter. The filter was placed in a Soxhlet type device and solvent cleaning of different polarity starts. Initially, the polymer was purified with methanol (250 mL) to remove any catalyst residues, then with acetone (250 mL) to remove any unreacted monomers, followed by hexane (250 mL) to remove any oligomers and finally with chloroform (250 mL) to extract the desired copolymer. The chloroform fraction was concentrated on a rotary evaporator to a volume of approximately 50 mL and the copolymer was precipitated in 800 mL of methanol. The precipitated copolymer was collected by vacuum filtration and dried for 24 h on a PTFE filter. Further drying of the polymer in a vacuum oven at 40 ◦C for 9 h led to a yield of 0.86 g of the final copolymer. The synthetic procedure and chemical structures of the synthesized conjugated copolymers are presented in the Supporting Information for clarification reasons (Scheme S1). The molecular characteristics of the copolymers were specified using GPC and the results are summarized in Table S1 (Supporting Information). The three different molecular weight copolymers are abbreviated as C1, C2 and C3 respectively as evident in Table S1 in the supporting information. In addition, in Figure S1 the GPC curves with respect to the different copolymers are presented.

#### *2.4. Synthesis of Graphene Oxide and Reduction to the Desired rGO*

Graphene oxide was synthesized with the modified Staudenmaier method which is widely established [2]. In a typical synthesis, powdered graphite (5 g, purum powder ≤ 0.2 mm; Fluka) was added to a mixture of concentrated sulfuric acid (200 mL, 95–97 wt%) and nitric acid (100 mL, 65 wt%) while cooling in an ice-water bath. Potassium chlorate powder (100 g, purum > 98.0%; Fluka) was added to the mixture gradually while stirring and cooling. The reaction was quenched after 18 h by pouring the mixture into distilled water and the oxidation product was washed until the pH reached a value of 6.0 and finally dried at room temperature. The resulting GO was re-dispersed in 200 mL distilled water under agitation for 24 h. Then, 50 mL of an NaBH<sup>4</sup> aqueous solution (8 mg/mL) were added and the dispersion was agitated for another 24 h [3]. Finally, the dispersion was centrifuged for five (5) times and the sediment was left to dry on a glass substrate. Using this procedure rGO was efficiently prepared.

#### *2.5. Preparation of the Hybrid Dispersions*

Many solvents have been proposed as appropriate dispersion mediums for rGO [27,28]. High-boiling point solvents showcase an outstanding performance in terms of stability. Our choice of dispersing rGO in o-DCB was two-fold: firstly to produce extremely stable dispersions of rGO and secondly it is a common solvent of conjugated polymers of the indaceno- type. Thus, we have prepared nine (9) different dispersions of hybrid materials using sonication for 6 h in each of the three different copolymers with 3, 5 and 10% wt rGO loading respectively as evident in Table 1.


**Table 1.** The oxidation and reduction potentials from the respective cyclic voltammograms for each system (recalibrated versus Fc/Fc<sup>+</sup> ) and the corresponding HOMO, LUMO and band gaps.


**Table 1.** *Cont.*

\* Number average molecular weights were calculated from Gel Permeation Chromatography measurements. (For additional information, refer to Supporting Information).

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

#### *3.1. Raman and X-ray Diffraction Analysis*

Raman spectroscopy was performed to study the oxidation of pristine graphite to GO and its further reduction to rGO. In Figure S2 in the Supporting Information the Raman spectra of the two allotropic forms of graphene oxide obtained are illustrated. In the GO spectrum, the two major peaks D and G are 1310 cm−<sup>1</sup> and 1590 cm−<sup>1</sup> respectively possess quite large deviations and structural deficiencies in relation to graphene due to the existence of the oxygen groups that alter sp<sup>2</sup> hybridization and therefore its structure. The imperfections are represented by the I<sup>D</sup> / I<sup>G</sup> ratio, which equals to 1.99, a value that is justified by the oxidation process of graphite. Correspondingly for the case of rGO, D and G peaks at 1301 cm−<sup>1</sup> and 1575 cm−<sup>1</sup> respectively show the structural defects from the non-selective removal of the oxygen groups due to the reduction which induces gaps along the structure. In this case, the ID/I<sup>G</sup> ratio increases to 2.23, indicating the successful rGO reduction [29].

Concerning the XRD measurements conducted on GO and rGO a clearcut difference on the first reflection is evident in the XRD spectra presented in Figure S3. Taking into consideration the primary reflection (001) and by applying Bragg's relationship one concludes that the reduction of the original oxygen groups leads to a change in the arrangement of the graphene sheets. Specifically, d(001) GO equals to 7.4 Å while d(001) rGO to 9.1 Å.

The successful chemical modification is verified by the increase of the neighboring graphite sheets distance as indicated by the synergy of literature [30] and experimental results. In Figure S3 the comparative diffraction diagram for GO and rGO is presented showcasing a distinct difference on the first reflection which reveals the larger interstitial space between the rGO sheets. The specific properties are desirable for the preparation of nanocomposite materials due to the capability of the conjugated polymer to be accommodated between the interstitial spaces of the laminar material.

TGA experiments were performed to study the thermal behavior of both the GO and the rGO. A minimal weight loss (8%) at approximately 100 ◦C is evident and corresponds to the water molecules, while functional groups (-O(CO)-, -OH, -O-) are decomposed at approximately 230 ◦C, leading to a second significant weight loss (30%). The graphitic lattice decomposition (50% weight loss) took place at approximately 500 ◦C (Figure S4A in the Supporting Information). The TGA studies on the rGO revealed a weight loss at approximately 10% due to the removal of moisture. The remaining functional groups (-O(CO)-, -OH, -O-) after the chemical modification are removed at approximately 190 ◦C leading to 20% weight loss which is less than the one observed in the GO further verifying the successful chemical modification. The graphitic lattice decomposition occurred at approximately 530 ◦C indicating higher thermal stability of the rGO due to the chemical modification of the functional groups (Figure S4B in the Supporting Information). The thermograph of the pristine conjugated copolymer (C3) and hybrid materials consisting of the conjugated copolymer C3 with different weight percentages of rGO are also presented in Figure S4C–F. The original decomposition of the pure conjugated copolymer took place at approximately 238 ◦C (Figure S4C). It is evident that the decomposition of the conjugated copolymer with 3 wt% of rGO initiated at 242 ◦C (Figure S4D), while for the 5 wt% rGO at 250 ◦C (Figure S4E) and for the 10% rGO at 260 ◦C (Figure S4F) indicating that the

higher the rGO ratio the greater the thermal stability of the final hybrid material. Coherent behavior was also observed in the remaining samples. Note that, the materials with the highest percentage of rGO not only showcased the highest stability but also less weight loss which is in accordance to previous studies [31]. that the higher the rGO ratio the greater the thermal stability of the final hybrid material. Coherent behavior was also observed in the remaining samples. Note that, the materials with the highest percentage of rGO not only showcased the highest stability but also less weight loss which is in accordance to previous studies [31].

#### *3.2. Morphological Characterization 3.2. Morphological Characterization*

*Polymers* **2022**, *14*, x FOR PEER REVIEW 6 of 12

approximately 10% due to the removal of moisture. The remaining functional groups (- Ο(CO)-, -OH, -O-) after the chemical modification are removed at approximately 190 °C leading to 20% weight loss which is less than the one observed in the GO further verifying the successful chemical modification. The graphitic lattice decomposition occurred at approximately 530 °C indicating higher thermal stability of the rGO due to the chemical modification of the functional groups (Figure S4B in the Supporting Information). The thermograph of the pristine conjugated copolymer (C3) and hybrid materials consisting of the conjugated copolymer C3 with different weight percentages of rGO are also presented in Figure S4C–F. The original decomposition of the pure conjugated copolymer took place at approximately 238 °C (Figure S4C). It is evident that the decomposition of the conjugated copolymer with 3 wt% of rGO initiated at 242 °C (Figure S4D), while for the 5 wt% rGO at 250 °C (Figure S4E) and for the 10% rGO at 260 °C (Figure S4F) indicating

The morphological characterization of GO, rGO and the final hybrid materials was performed through SEM measurements. In Figure 1 the difference in the nanosheets of GO (Figure 1A) and rGO (Figure 1B) in the dispersion form (an aqueous dispersion for GO and an o-DCB dispersion for rGO which was left to dry at an elevated temperature overnight under vacuum) is depicted. The SEM micrographs presented in Figure 1C,D correspond to the solid form of the GO and the rGO respectively, indicating an apparent introduction of abnormalities in the rGO morphology compared to the defect-free GO, which are attributed to the reduction process as vacancies that occur in the lattice during the removal of the oxygen groups. From the SEM images it is evident that the conversion of the graphite morphology to graphene oxide was accomplished successfully maintaining its lamellar formation. Also, it is straightforward that the film and solid forms exhibit significant alternations due to the re-dispersion of GO and rGO in o-DCB for the case of film state. The morphological characterization of GO, rGO and the final hybrid materials was performed through SEM measurements. In Figure 1 the difference in the nanosheets of GO (Figure 1A) and rGO (Figure 1B) in the dispersion form (an aqueous dispersion for GO and an o-DCB dispersion for rGO which was left to dry at an elevated temperature overnight under vacuum) is depicted. The SEM micrographs presented in Figure 1C,D correspond to the solid form of the GO and the rGO respectively, indicating an apparent introduction of abnormalities in the rGO morphology compared to the defect-free GO, which are attributed to the reduction process as vacancies that occur in the lattice during the removal of the oxygen groups. From the SEM images it is evident that the conversion of the graphite morphology to graphene oxide was accomplished successfully maintaining its lamellar formation. Also, it is straightforward that the film and solid forms exhibit significant alternations due to the re-dispersion of GO and rGO in o-DCB for the case of film state.

**Figure 1.** SEM images of the GO and rGO in film (**A**,**B**) and in solid (**C**,**D**) forms*.* **Figure 1.** SEM images of the GO and rGO in film (**A**,**B**) and in solid (**C**,**D**) forms.

In Figure 2 the SEM micrograph in conjunction with the elemental mapping analysis of the hybrid material which is constituted by copolymer 2 (C2) with 5 wt% rGO is demonstrated. The interaction of the conjugated copolymer with the rGO due to π-π stacking led In Figure 2 the SEM micrograph in conjunction with the elemental mapping analysis of the hybrid material which is constituted by copolymer 2 (C2) with 5 wt% rGO is demonstrated. The interaction of the conjugated copolymer with the rGO due to π-π stacking led to the intercalation of the copolymer between the graphene nanosheets, a behavior that has been reported for similar layered systems [32] (Figure 2A). This is further supported by the EDX in which the elemental mapping analysis confirmed the existence of carbon (Figure 2B) (a common element of the hybrid system), sulfur (Figure 2C) which is derived from the thiophene groups in the copolymer structural unit and oxygen (Figure 2D), which is solely found in rGO and verifies the successful preparation of the hybrid material. The SEM/EDX data obtained from the different copolymers using 3, 5 and 10 wt% of rGO, showcased similar results, suggesting the non-covalent attachment of the rGO on the conjugated copolymers even in low concentration values.

to the intercalation of the copolymer between the graphene nanosheets, a behavior that has been reported for similar layered systems [32] (Figure 2A). This is further supported by the EDX in which the elemental mapping analysis confirmed the existence of carbon (Figure 2B) (a common element of the hybrid system), sulfur (Figure 2C) which is derived from the thiophene groups in the copolymer structural unit and oxygen (Figure 2D), which is solely found in rGO and verifies the successful preparation of the hybrid mate-

the conjugated copolymers even in low concentration values.

**Figure 2.** SEM images of a hybrid system with the Copolymer 2 and the rGO (**A**). There is an apparent intercalation that is supported with the aid of EDS, where the elemental mapping analysis confirms the existence of Carbon (**B**), Sulfur (**C**) and Oxygen (**D**). **Figure 2.** SEM images of a hybrid system with the Copolymer 2 and the rGO (**A**). There is an apparent intercalation that is supported with the aid of EDS, where the elemental mapping analysis confirms the existence of Carbon (**B**), Sulfur (**C**) and Oxygen (**D**).

Interestingly enough, organic cells-related applications are based on polymer films and therefore the morphological characterization of the hybrid material which is comprised by the C2 copolymer with either 5 wt% or 10 wt% rGO were conducted in thin film state. The spin coated samples were approximately ~100 nm thick and the SEM studies indicated similar structures to the ones obtained during bulk characterization. The results are promising for photovoltaic applications, but more comprehensive studies are required. Two representative SEM micrographs are presented in the Supporting Infor-Interestingly enough, organic cells-related applications are based on polymer films and therefore the morphological characterization of the hybrid material which is comprised by the C2 copolymer with either 5 wt% or 10 wt% rGO were conducted in thin film state. The spin coated samples were approximately ~100 nm thick and the SEM studies indicated similar structures to the ones obtained during bulk characterization. The results are promising for photovoltaic applications, but more comprehensive studies are required. Two representative SEM micrographs are presented in the Supporting Information (Figure S5a,b).

#### *3.3. Optoelectronic Properties*

mation (Figure S5a,b).

*3.3. Optoelectronic Properties* In Figure 3 the UV-Vis spectra obtained from 300–900 nm for the three pristine copolymer systems and the hybrid materials with different rGO concentration are given. For copolymer 1 (C1) (Figure 3A) two distinct peaks at 423 nm and 723 nm can be clearly identified. With the addition of rGO, the first absorption peak was shifted from 423 nm to 429 nm while the second peak from 723 nm to 729 nm. This phenomenon indicates that even a relatively small (3% wt) addition of rGO is able to alter the absorption maxima of the copolymer and hence its energy gap. This phenomenon is also observed in samples with higher rGO concentration, which also exhibit similar displacements at a higher absorption intensity. Consistently, similar results were recorded for copolymer C2 (Figure 3B) which exhibits three absorption maxima (637 nm, 411 nm and 319 nm) but the shift In Figure 3 the UV-Vis spectra obtained from 300–900 nm for the three pristine copolymer systems and the hybrid materials with different rGO concentration are given. For copolymer 1 (C1) (Figure 3A) two distinct peaks at 423 nm and 723 nm can be clearly identified. With the addition of rGO, the first absorption peak was shifted from 423 nm to 429 nm while the second peak from 723 nm to 729 nm. This phenomenon indicates that even a relatively small (3% wt) addition of rGO is able to alter the absorption maxima of the copolymer and hence its energy gap. This phenomenon is also observed in samples with higher rGO concentration, which also exhibit similar displacements at a higher absorption intensity. Consistently, similar results were recorded for copolymer C2 (Figure 3B) which exhibits three absorption maxima (637 nm, 411 nm and 319 nm) but the shift only occurs for two maxima, since the peak at 637 nm shifts to 640 nm and the peak at 319 nm shifts to 322 nm. Finally, copolymer C3 (Figure 3C) demonstrates a similar shift in wavelength as C1 (from 713 nm to 719 nm and from 434 nm to 440 nm), thus reaffirming the impact of the rGO addition on the optical properties.

only occurs for two maxima, since the peak at 637 nm shifts to 640 nm and the peak at 319

the impact of the rGO addition on the optical properties.

**Figure 3.** UV-Vis spectra of the: (**A**) C1 systems; (**B**) C2 systems; and (**C**) C3 systems. Distinct wavelength shifts can be observed in every system for even a minimum rGO addition, at (red line) C-3% rGO, (blue line) C-5% rGO, and (purple line) C-10% rGO. **Figure 3.** UV-Vis spectra of the: (**A**) C1 systems; (**B**) C2 systems; and (**C**) C3 systems. Distinct wavelength shifts can be observed in every system for even a minimum rGO addition, at (red line) C-3% rGO, (blue line) C-5% rGO, and (purple line) C-10% rGO.

Cyclic voltammetry was performed to correlate the oxidation and the reduction potential onsets with the respective HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital) levels as it has been previously described for such conjugated copolymers by our group [33]. The cyclic voltammograph of ferrocene in acetonitrile (ACN) was used as reference for the copolymers and is presented in the Supporting

Information (Figure S6). The oxidation and reduction potentials were recalibrated versus the Fc/Fc<sup>+</sup> redox couple and the corresponding HOMO and LUMO levels were calculated using the following equations: in acetonitrile (ACN) was used as reference for the copolymers and is presented in the Supporting Information (Figure S6). The oxidation and reduction potentials were recalibrated versus the Fc/Fc<sup>+</sup> redox couple and the corresponding HOMO and LUMO levels for such conjugated copolymers by our group [33]. The cyclic voltammograph of ferrocene in acetonitrile (ACN) was used as reference for the copolymers and is presented in the Supporting Information (Figure S6). The oxidation and reduction potentials were recali-

were calculated using the following equations:

*Polymers* **2022**, *14*, x FOR PEER REVIEW 9 of 12

*Polymers* **2022**, *14*, x FOR PEER REVIEW 9 of 12

$$\mathbf{E\_{HOMO}} = \mathbf{5.1} + \mathbf{E^{ox}}\_{\text{onset}} \tag{1}$$

brated versus the Fc/Fc<sup>+</sup> redox couple and the corresponding HOMO and LUMO levels

Cyclic voltammetry was performed to correlate the oxidation and the reduction potential onsets with the respective HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital) levels as it has been previously described for such conjugated copolymers by our group [33]. The cyclic voltammograph of ferrocene

Cyclic voltammetry was performed to correlate the oxidation and the reduction potential onsets with the respective HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital) levels as it has been previously described

$$\text{E}\_{\text{LUMO}} = \text{5.1} + \text{E}^{\text{red}}\_{\text{onset}} \tag{2}$$

The cyclic voltammographs (CVs) for the pristine C1 copolymer and the produced hybrid materials after the addition of 3, 5 and 10% wt rGO respectively are illustrated in Figure 4. It is apparent that for lower rGO concentrations (3 and 5 wt%) a significant alteration on the redox potentials is experienced and therefore to the HOMO and LUMO levels, while in higher concentration (10 wt%) the redox potentials are similar to those obtained for the pristine copolymer [34]. This effect is observed for all three hybrid systems since it is also evident in Figure 5 (C2) and in Figure 6 (C3). Table 1 summarizes the results provided from the CVs as well as the calculated HOMO, LUMO levels and the electrochemical band gap (Eg). The cyclic voltammographs (CVs) for the pristine C1 copolymer and the produced hybrid materials after the addition of 3, 5 and 10% wt rGO respectively are illustrated in Figure 4. It is apparent that for lower rGO concentrations (3 and 5 wt%) a significant alteration on the redox potentials is experienced and therefore to the HOMO and LUMO levels, while in higher concentration (10 wt%) the redox potentials are similar to those obtained for the pristine copolymer [34]. This effect is observed for all three hybrid systems since it is also evident in Figure 5 (C2) and in Figure 6 (C3). Table 1 summarizes the results provided from the CVs as well as the calculated HOMO, LUMO levels and the electrochemical band gap (Eg). ELUMO = 5.1 + Eredonset (2) The cyclic voltammographs (CVs) for the pristine C1 copolymer and the produced hybrid materials after the addition of 3, 5 and 10% wt rGO respectively are illustrated in Figure 4. It is apparent that for lower rGO concentrations (3 and 5 wt%) a significant alteration on the redox potentials is experienced and therefore to the HOMO and LUMO levels, while in higher concentration (10 wt%) the redox potentials are similar to those obtained for the pristine copolymer [34]. This effect is observed for all three hybrid systems since it is also evident in Figure 5 (C2) and in Figure 6 (C3). Table 1 summarizes the results provided from the CVs as well as the calculated HOMO, LUMO levels and the

**Figure 4.** Cyclic voltammograms of the C1 systems for both the oxidation and the reduction process. in 0.1 mol L<sup>−</sup><sup>1</sup> Bu4NBF4. Scan rate 0.050 V s−1. Signals presented as: (red line) C1-3% rGO, (blue line) C1-5% rGO, and (purple line) C1-10% rGO. **Figure 4.** Cyclic voltammograms of the C1 systems for both the oxidation and the reduction process. in 0.1 mol L−<sup>1</sup> Bu4NBF<sup>4</sup> . Scan rate 0.050 V s−<sup>1</sup> . Signals presented as: (red line) C1-3% rGO, (blue line) C1-5% rGO, and (purple line) C1-10% rGO. **Figure 4.** Cyclic voltammograms of the C1 systems for both the oxidation and the reduction process. in 0.1 mol L<sup>−</sup><sup>1</sup> Bu4NBF4. Scan rate 0.050 V s−1. Signals presented as: (red line) C1-3% rGO, (blue line) C1-5% rGO, and (purple line) C1-10% rGO.

in 0.1 mol L−1 Bu4NBF4. Scan rate 0.050 V s−1. Signals presented as: (red line) C3-3% rGO, (blue line) C3-5% rGO, and (purple line) C3-10% rGO. **Figure 5.** Cyclic voltammograms of the C2 systems for both the oxidation and the reduction process. in 0.1 mol L−1 Bu4NBF4. Scan rate 0.050 V s−1. Signals presented as: (red line) C3-3% rGO, (blue line) C3-5% rGO, and (purple line) C3-10% rGO. **Figure 5.** Cyclic voltammograms of the C2 systems for both the oxidation and the reduction process. in 0.1 mol L−<sup>1</sup> Bu4NBF<sup>4</sup> . Scan rate 0.050 V s−<sup>1</sup> . Signals presented as: (red line) C3-3% rGO, (blue line) C3-5% rGO, and (purple line) C3-10% rGO.

**Figure 6.** Cyclic voltammograms of the C3 systems for both the oxidation and the reduction process. in 0.1 mol L−1 Bu4NBF4. Scan rate 0.050 V s−1 . **Figure 6.** Cyclic voltammograms of the C3 systems for both the oxidation and the reduction process. in 0.1 mol L−<sup>1</sup> Bu4NBF<sup>4</sup> . Scan rate 0.050 V s−<sup>1</sup> .

#### **4. Conclusions 4. Conclusions**

Graphene oxide (GO) and reduced graphene oxide (rGO) were synthesized and successfully combined with indacenodithiophene- and indacenodithienothiophene-based conjugated copolymers for the preparation of hybrid materials in order to study the alteration on their optoelectronic properties due to non-covalent binding (π-π stacking) with rGO. The conjugated copolymers were synthesized through Stille aromatic coupling. Staudenmaier reaction was utilized for the synthesis of GO, which was further reduced for the preparation of rGO. The conjugated polymers were mixed using different ratios of rGO and were ultrasonicated in ortho-dichlorobenzene that constitutes an excellent dispersion medium for each system, to produce stable dispersions. To identify the successful synthesis of GO and rGO, morphological and structural characterization was performed using Scanning Electron Microscopy (SEM), Raman spectroscopy and X-Ray Diffraction analysis. UV-Vis absorption spectrophotometry was used to determine the optical properties of the pristine copolymers and the hybrid materials. The experiments indicated a significant wavelength shift in the absorption maxima for all rGOs of the order of 3–6 nm, which justifies the alteration and fine-tuning of the optical properties of the copolymers with the aid of rGO. Finally, electrochemical characterization was performed via Cyclic Voltammetry (CV) to determine the energy levels of copolymers and mixtures. From the determination of oxidation and reduction potentials and the calculated HOMO and LUMO levels it is concluded that small percentages of rGO (3–5 wt%) can alter the energy levels of the copolymers and ultimately decrease the electrochemical Energy gap (Eg), while larger concentrations of rGO can return these attributes to their original state. This suggests that the specific hybrid materials are quite promising for applications such as organic solar cells, where a tunable band gap and wavelength are of strategic importance. Graphene oxide (GO) and reduced graphene oxide (rGO) were synthesized and successfully combined with indacenodithiophene- and indacenodithienothiophene-based conjugated copolymers for the preparation of hybrid materials in order to study the alteration on their optoelectronic properties due to non-covalent binding (π-π stacking) with rGO. The conjugated copolymers were synthesized through Stille aromatic coupling. Staudenmaier reaction was utilized for the synthesis of GO, which was further reduced for the preparation of rGO. The conjugated polymers were mixed using different ratios of rGO and were ultrasonicated in ortho-dichlorobenzene that constitutes an excellent dispersion medium for each system, to produce stable dispersions. To identify the successful synthesis of GO and rGO, morphological and structural characterization was performed using Scanning Electron Microscopy (SEM), Raman spectroscopy and X-Ray Diffraction analysis. UV-Vis absorption spectrophotometry was used to determine the optical properties of the pristine copolymers and the hybrid materials. The experiments indicated a significant wavelength shift in the absorption maxima for all rGOs of the order of 3–6 nm, which justifies the alteration and fine-tuning of the optical properties of the copolymers with the aid of rGO. Finally, electrochemical characterization was performed via Cyclic Voltammetry (CV) to determine the energy levels of copolymers and mixtures. From the determination of oxidation and reduction potentials and the calculated HOMO and LUMO levels it is concluded that small percentages of rGO (3–5 wt%) can alter the energy levels of the copolymers and ultimately decrease the electrochemical Energy gap (Eg), while larger concentrations of rGO can return these attributes to their original state. This suggests that the specific hybrid materials are quite promising for applications such as organic solar cells, where a tunable band gap and wavelength are of strategic importance.

**Supplementary Materials:** The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Scheme S1: Chemical reactions and structures of: (**A**) indacenothiophenealt -3,6-bis(5-bromothiophen-2-yl)-2,5-bis(2-octyldodecyl)pyrrolo [3,4-c]pyrrole (IDT and Dibromo-DPP) or C1; (**B**) indacenothiophene -alt-4,7-dibromo-5,6-difluorobenzo[1,2,5]thiadiazole (IDT and difluoro-BTD) or C2; and (**C**) Indacenodithienothiophene-alt-3,6-bis(5-bromothieno[3,2-b]thiophen-2-yl)-2,5 bis(2octyldodecyl)pyrrolo[3,4-c]pyrrole (IDTT and Dibromo-DPP) or C3 copolymers; Figure S1: Gel Permeation Chromatography (GPC) curves of the three different copolymers where the black line corresponds to the C1 copolymer, the red line to the C2 copolymer and the blue line to the C3; Table S1: Molecular characteristics of the three different copolymers as directly calculated from gel permission chromatography; Figure S2: Raman Spectra obtained for GO and rGO. Both spectra represent materials with a great number of defects which is evident by the respective IG/I<sup>D</sup> ratio. The red curve corresponds to the rGO while the black to the GO; Figure S3: XRD measurements for GO and rGO. The red curve corresponds to the rGO while the black to the GO; Figure S4: TGA thermograms under nitrogen atmosphere corresponding to: (**A**) GO; (**B**) rGO; (**C**) C3 copolymer; (**D**) C3 copolymer with 3 wt% rGO; (**E**) C3 copolymer with 5 wt% rGO and F) C3 copolymer **Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/polym14235292/s1, Scheme S1: Chemical reactions and structures of: (**A**) indacenothiophene-alt -3,6-bis(5-bromothiophen-2-yl)-2,5-bis(2-octyldodecyl)pyrrolo [3,4-c]pyrrole (IDT and Dibromo-DPP) or C1; (**B**) indacenothiophene -alt-4,7-dibromo-5,6-difluorob enzo[1,2,5]thiadiazole (IDT and difluoro-BTD) or C2; and (**C**) Indacenodithienothiophene-alt-3,6 bis(5-bromothieno[3,2-b]thiophen-2-yl)-2,5 bis(2octyldodecyl)pyrrolo[3,4-c]pyrrole (IDTT and Dibromo-DPP) or C3 copolymers; Figure S1: Gel Permeation Chromatography (GPC) curves of the three different copolymers where the black line corresponds to the C1 copolymer, the red line to the C2 copolymer and the blue line to the C3; Table S1: Molecular characteristics of the three different copolymers as directly calculated from gel permission chromatography; Figure S2: Raman Spectra obtained for GO and rGO. Both spectra represent materials with a great number of defects which is evident by the respective IG/I<sup>D</sup> ratio. The red curve corresponds to the rGO while the black to the GO; Figure S3: XRD measurements for GO and rGO. The red curve corresponds to the rGO while the black to the GO; Figure S4: TGA thermograms under nitrogen atmosphere corresponding to: (**A**) GO; (**B**) rGO; (**C**) C3 copolymer; (**D**) C3 copolymer with 3 wt% rGO; (**E**) C3 copolymer with

with 10 wt% rGO; Figure S5: SEM images corresponding to the C2 copolymer with (**A**) 5 wt% rGO;

5 wt% rGO and F) C3 copolymer with 10 wt% rGO; Figure S5: SEM images corresponding to the C2 copolymer with (**A**) 5 wt% rGO; and (**B**) 10 wt% rGO. The samples were prepared using the spin coating technique leading to thicknesses of approximately 100 nm; Figure S6: Cyclic voltammogram of ferrocene in ACN which was used as reference for the copolymers

**Author Contributions:** Conceptualization, C.L.C. and A.A.; methodology, C.L.C. and A.A.; validation, A.C.L., A.K., M.S., C.L.C., V.G.G. and A.A.; formal analysis, A.C.L., A.K., M.S., G.-M.M., I.M., D.V.V. and D.M.; investigation, A.C.L., A.K. and M.S.; resources, D.A.I., C.L.C., V.G.G. and A.A.; data curation, A.C.L., A.K., M.S., G.-M.M., I.M., D.M., C.G. and M.A.K.; writing—original draft preparation, A.C.L., C.L.C. and A.A.; writing—review and editing, A.C.L., A.K., G.-M.M., I.M., D.A.I., C.L.C. and A.A.; visualization, C.L.C. and A.A.; supervision, C.L.C., V.G.G. and A.A.; funding acquisition, D.A.I. and A.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by [the Ministry of Science and Higher Education of the Russian Federation within State Contract], grant no. [075-15-2022-1105].

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

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

#### **References**


**Maria Karayianni , Dimitra Koufi and Stergios Pispas \***

Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, 116 35 Athens, Greece

**\*** Correspondence: pispas@eie.gr

**Abstract:** The electrostatic complexation between double hydrophilic block copolymers (DHBCs) and a model porphyrin was explored as a means for the development of polyion complex micelles (PICs) that can be utilized as photosensitive porphyrin-loaded nanoparticles. Specifically, we employed a poly(2-(dimethylamino) ethyl methacrylate)-*b*-poly[(oligo ethylene glycol) methyl ether methacrylate] (PDMAEMA-*b*-POEGMA) diblock copolymer, along with its quaternized polyelectrolyte copolymer counterpart (QPDMAEMA-*b*-POEGMA) and 5,10,15,20-tetraphenyl-21H,23Hporphine-p,p0 ,p",p 000-tetrasulfonic acid tetrasodium hydrate (TPPS) porphyrin. The (Q)PDMAEMA blocks enable electrostatic binding with TPPS, thus forming the micellar core, while the POEGMA blocks act as the corona of the micelles and impart solubility, biocompatibility, and stealth properties to the formed nanoparticles. Different mixing charge ratios were examined aiming to produce stable nanocarriers. The mass, size, size distribution and effective charge of the resulting nanoparticles, as well as their response to changes in their environment (i.e., pH and temperature) were investigated by dynamic and electrophoretic light scattering (DLS and ELS). Moreover, the photophysical properties of the complexed porphyrin along with further structural insight were obtained through UV-vis (200-800 nm) and fluorescence spectroscopy measurements.

**Keywords:** double hydrophilic block copolymers; porphyrins; polyion complex micelles; photosensitizers

#### **1. Introduction**

Successful cancer treatment is one of the most sought-after goals of modern-day medicine, since cancer remains a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020 according to the World Health Organization [1]. Among the various proposed therapeutic strategies, photodynamic therapy (PDT) holds significant promise owing to its efficiency, site-specificity, and noninvasive characteristics. It is being used not only for the treatment of tumors related to various types of cancers (e.g., skin, esophageal, lung) but also a number of other diseases and medical conditions, such as atherosclerosis, rheumatoid arthritis, macular degeneration, psoriasis, and acne [2]. The working principle of PDT is based on the administration of photosensitizers that accumulate in pathological tissue and are subsequently exposed to a light source with appropriate wavelength so as to generate the production of reactive oxygen species (ROS), which in turn cause cell death [3–6]. Due to its selective action, it causes minimal damage to the surrounding healthy tissue, has no severe local or systemic side effects, is not painful, is well tolerated by patients, allows for outpatient use, and can even be applied in parallel with other therapeutic protocols [2,3]. All these advantages in combination with documented good therapeutic results render PDT a widespread contemporary method for the treatment of cancer and other infectious diseases.

Evidently, the role of the photosensitizer is of utmost importance for the effective application of PTD. Having entered the cell, the photoactive molecule is appropriately irradiated, leading to its excitation from the ground to the excited singlet state as a result

**Citation:** Karayianni, M.; Koufi, D.; Pispas, S. Development of Double Hydrophilic Block Copolymer/ Porphyrin Polyion Complex Micelles towards Photofunctional Nanoparticles. *Polymers* **2022**, *14*, 5186. https://doi.org/10.3390/ polym14235186

Academic Editor: Nikolaos Politakos

Received: 31 October 2022 Accepted: 22 November 2022 Published: 29 November 2022

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

**Copyright:** © 2022 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/).

of the photon absorption [3–5]. Next, the excited photosensitizers either relax back to the ground state via fluorescence photon emission or are transformed to the excited triplet state, whose energy under well-oxygenated conditions can be transferred to the surrounding tissue oxygen molecules, mainly producing singlet oxygen (1O2). These singlet oxygen particles are characterized by extremely strong oxidizing properties and can destroy tumor cells, either directly by inducing apoptotic and/or necrotic cell death or indirectly by provoking autophagy [3,4]. Over the years of PDT clinical practice, several characteristics have been established as prerequisites for ideal photosensitizers. These include high chemical purity, stability at room temperature, high photochemical reactivity, minimum dark cytotoxicity, preferential retention at the targeted tissue, facile solubility in bodily fluids, low cost, and wide availability, among others [2,3]. Of course, the most widely used photosensitizers consist of porphyrins (a class of heterocyclic macrocycle organic compounds) and their derivatives, since the historic discovery of hematoporphyrin and its photo-related medical properties in the beginning of the previous century [2].

Owing to their hydrophobic nature, porphyrins tend to self-assemble in aqueous media, forming aggregates of intriguing structures and unique features [7]. Nevertheless, aggregation reduces their photoactivity and thus therapeutic efficiency. In order to enhance porphyrin water solubility, as well as circulation lifetime and tumor specificity, and at the same time reduce aspecific tissue accumulation, the use of various types of polymeric nanocarriers has been proposed [4–6,8]. Among the plethora of available macromolecular architectures, the use of double hydrophilic block copolymers (DHBCs)—consisting of one charged and one hydrophilic block—as delivery vehicles is of particular interest due to their interesting self-assembly behavior [9–12]. This kind of copolymer is able to interact with oppositely charged bioactive species, forming polyion complex micelles (PICs) where the interacting charged species form the micellar core and the hydrophilic blocks the corona, thus providing solubility or even biocompatibility and stealth properties to the formed nanoparticles. This principle has been extensively exploited over the years as a means of developing porphyrin-loaded micelles for potential PDT applications. Starting with the work of Kataoka (a pioneer in PICs) and his coworkers on dendritic zinc porphyrins [13,14], followed by the extended studies of the Shi group on 5,10,15,20-tetrakis- (4-sulfonatophenyl)-porphyrin (TPPS) and its metal ions [15–20], as well as numerous other similar investigations involving not only DHBC [21–26] but also surfactants [27,28], polycations [29], nonionic triblock copolymers [30], or even polymeric membranes [31], one can only begin to fathom the importance of these systems considering their prospective therapeutic capability, and thus justify the ongoing considerable scientific interest they have attracted.

In this work, we report on the formation of photofunctional nanoparticles (owing to the intrinsic properties of the porphyrin) through the electrostatic complexation between the poly(2-(dimethylamino) ethyl methacrylate)-*b*-poly[(oligo ethylene glycol) methyl ether methacrylate] (PDMAEMA-*b*-POEGMA) double hydrophilic block copolymer or its quaternized strong polyelectrolyte counterpart (QPDMAEMA-*b*-POEGMA) and 5,10,15,20-tetraphenyl-21H,23H-porphine-p,p0 ,p",p000-tetrasulfonic acid tetrasodium hydrate (TPPS) porphyrin. The resulting PIC micelles comprise a mixed polyelectrolyte– porphyrin (Q)PDMAEMA/TPPS core and a hydrophilic biocompatible POEGMA corona/ shell. Their solution properties in regard to their mass, size, size distribution and effective charge at varying mixing ratios with increasing porphyrin content were thoroughly investigated by means of dynamic and electrophoretic light-scattering techniques. In parallel, UV-vis and fluorescence detailed spectroscopic studies were conducted to probe the photophysical characteristics of the complexed porphyrin, extract additional information regarding its morphology and aggregation state, and of course validate the photosensitivity of the produced nanoparticles. The pH-dependent charge density of the PDMAEMA polyelectrolyte and aggregation behavior of TPPS porphyrin necessitated the investigation to be performed at different pH values, namely, pH 7 and 3. Moreover, the effect of temperature on the overall properties of the already formed PICs was examined.

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

### *2.1. Materials*

The 5,10,15,20-tetraphenyl-21H,23H-porphine-p,p0 ,p",p000-tetrasulfonic acid tetrasodium hydrate (TPPS) porphyrin (*M*<sup>w</sup> = 1022.92 g/mol, anhydrous basis) (Scheme 1a) and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as received.

**Scheme 1.** Molecular structures of (**a**) TPPS porphyrin, (**b**) PDMAEMA-*b*-POEGMA copolymer and (**c**) its quaternized counterpart QPDMAEMA-*b*-POEGMA.

#### *2.2. Block Copolymer Synthesis and Quaternization*

The synthetic procedure of the PDMAEMA-*b*-POEGMA block copolymer by means of reversible addition fragmentation chain transfer (RAFT) polymerization, as well as the subsequent quaternization reaction, were performed in a similar manner to the one described in detail in a previous publication [32]. In brief, the synthetic route comprises two steps, starting with the initial polymerization of the DMAEMA monomer under appropriate conditions and the formation of the PDMAEMA homopolymer, which then acts as a macrochain transfer agent (macro-CTA) for the polymerization of the OEGMA monomer (which bears 9 ethylene glycol units) and the formation of the POEGMA block. The molecular characteristics of the resulting copolymer as determined by SEC and <sup>1</sup>H NMR are summarized in Table 1, while its molecular structure is shown in Scheme 1b. The quaternization of the PDMAEMA-*b*-POEGMA precursor was performed by appropriate reaction with methyl iodide (CH3I), thus transforming the tertiary amines of the PDMAEMA block to quaternary ammonium groups, as seen in Scheme 1c. The corresponding molecular characteristics are also presented in Table 1.

**Table 1.** Molecular characteristics of PDMAEMA-*b*-POEGMA and QPDMAEMA-*b*-POEGMA copolymers.


<sup>a</sup> Determined by SEC; <sup>b</sup> determined by <sup>1</sup>H NMR; <sup>c</sup> calculated based on the *M*<sup>n</sup> of the corresponding monomeric units; <sup>d</sup> calculated assuming 100% quaternization and the composition of the precursor copolymer.

#### *2.3. Preparation of the Polyion Complex Micelles (PICs)*

Initially, stock solutions of the PDMAEMA-*b*-POEGMA copolymer or its quaternized counterpart QPDMAEMA-*b*-POEGMA at a concentration of 1 mg/mL and TPPS porphyrin at 0.53 mg/mL in water for injection (WFI) were prepared (magnetic stirring was usually employed in order to assist solubilization) and left overnight at ambient conditions to equilibrate. The pH of these solutions was around 6.5, henceforth called pH 7 in the following. For the formation of the complexes, appropriate aliquots, namely, 0.4, 1, 1.4, and 2 mL, of the porphyrin solution were added to 2 mL of the copolymer solution under stirring, mixing of the two constituent solutions was continued for 5 min, and at the final stage, dilution with WFI to a total volume of 10 mL was performed. In this way, the copolymer concentration is kept constant throughout the series of complex solutions, while porphyrin concentration increases thus changing the molar ratio of the two components. The resulting solutions were stable and no precipitation was observed. The stock solution concentration and mixing ratios were suitably chosen so as the molar ratio of the TPPS sulfonate groups to the corresponding tertiary amine groups of the DHBC or quaternary ammonium groups of its quaternized counterpart (QDHBC)—denoted as SO<sup>3</sup> −/NR<sup>2</sup> or NR<sup>3</sup> + , where R stands for the methyl group—ranged from 20% to 100%. The characteristics of the complex solutions in regard to the final concentration of the components and charged groups ratio are given in Table 2. Exactly the same procedure was followed for the preparation of the corresponding complexes at pH 3, starting with copolymer and porphyrin stock solutions whose pH was adjusted to 3 by appropriate addition of 0.1 M HCl, leading again to stable complex solutions.

**Table 2.** Characteristics of the complex solutions of the DHBC or the QDHBC and TPPS porphyrin.


<sup>a</sup> R = CH3.

#### *2.4. Methods*

*Dynamic Light Scattering (DLS).* DLS measurements were performed on an ALV/CGS-3 compact goniometer system (ALVGmbH, Hessen, Germany) equipped with an ALV-5000/EPP multi-tau digital correlator, a He-Ne laser (*λ* = 632.8 nm), and an avalanche photodiode detector. All sample solutions were filtered through 0.45 µm hydrophilic PVDF syringe filters (ALWSCI Group, Hangzhou, China) before measurement in order to remove any dust particles or large aggregates. The samples were loaded into standard 1 cm width soda-lime glass dust-free cylindrical cells and measurements were performed at a series of angles in the range of 45–135◦ . A circulating water bath was used to set the temperature at 25 ◦C, or at the desired value (25–60 ◦C) in the case of temperaturedependent measurements.

The measured normalized time autocorrelation functions of the scattered light intensity *g*<sup>2</sup> (*t*) were fitted with the aid of the CONTIN analysis, thus obtaining the distribution of relaxation times *τ*. Assuming that the observed fluctuations of the scattered intensity are caused by diffusive motions, the apparent diffusion coefficient *D*app is related to the relaxation time *τ* as *D*app = 1/*τq* 2 , where *q* is the scattering vector defined as 4*πn*<sup>0</sup> sin(*θ*/2)/*λ*<sup>0</sup> with *n*0, *θ* and *λ*<sup>0</sup> the solvent refractive index, the scattering angle, and the wavelength of the laser in vacuum, respectively. From the apparent diffusion coefficient *D*app, the hydrodynamic radius *R*<sup>h</sup> can be obtained using the Stokes–Einstein relationship *R<sup>h</sup>* = *kBT*/6*πη*0*Dapp*, where *k*<sup>B</sup> is the Boltzmann constant, *T* is the temperature, and *η*<sup>0</sup> is the viscosity of the solvent [33,34].

*Electrophoretic Light Scattering (ELS).* Zeta potential (*ζ*P) measurements were conducted with a Zetasizer Nano-ZS (Malvern Panalytical Ltd., Malvern, United Kingdom) equipped with a He-Ne laser (*λ* = 633 nm) and an avalanche photodiode detector. The Henry equation in the Smoluchowski approximation was used for zeta potential calculation [35]. *ζ*<sup>P</sup> values were determined using the Smoluchowski equation *ζ<sup>P</sup>* = 4*πηυ*/*ε*, where *η* is the solvent viscosity, *υ* the electrophoretic mobility, and *ε* the dielectric constant of the solvent, and are reported as averages of fifty repeated measurements at a 13◦ scattering angle and room temperature.

*UV-vis spectroscopy.* UV-vis absorption spectra of the complexes and neat porphyrin and copolymer solutions were recorded between 200 and 800 nm wavelength using a UV-vis NIR double-beam spectrophotometer (Lambda 19 by Perkin Elmer, Waltham, MA, USA) at room temperature. Appropriate dilutions of the samples were performed so as not to exceed maximum acceptable absorbance values. Specifically, the complex solutions were diluted with a factor 1:20 and the neat TPPS 1:100 (final concentration 5.3 µg/mL).

*Fluorescence spectroscopy.* Steady-state fluorescence spectra of the neat and complexed TPPS porphyrin were recorded with a double-grating excitation and a single-grating emission spectrofluorometer (Fluorolog-3, model FL3-21, Jobin Yvon-Spex, Horiba Ltd., Kyoto, Japan) at either room temperature or higher temperatures regulated by a circulating water bath. Excitation wavelength used was *λ* = 515 nm and emission spectra were recorded in the region 535–800 nm, with an increment of 1 nm, using an integration time of 0.1 s, and slit openings of 2 nm for both the excitation and the emitted beam. Under the employed experimental conditions, fluorescence from the TPPS was observed and utilized to extract information regarding its structure, while the neat copolymer solutions did not show any significant fluorescence.

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

#### *3.1. PDMAEMA-b-POEGMA and TPPS Complexation*

At first, we investigated the formation of PICs through the electrostatic complexation between the PDMAEMA-*b*-POEGMA DHBC and TPPS porphyrin at both pH 7 and 3. The pH value plays a significant role in the protonation state of both the tertiary amine groups of the DHBC, which become fully protonated at pH 3, thus increasing charge density, as well as the pyrrolic nitrogen atoms of the porphyrin molecule that are protonated at pH below 5, thus transforming the porphyrin from its free-base form (H2TPPS4−) to its diacid form (H4TPPS2−) [15–17]. The solution properties of the stable complex solutions were initially examined by means of DLS measurements, and Figure 1 shows the obtained scattering intensity values derived from measurements at 90◦ (*I*90) as a function of the porphyrin concentration (*C*TPPS) or equivalently charged groups ratio (SO<sup>3</sup> −/NR2, R = CH3) at both pH values. Note that the *I*<sup>90</sup> values at *C*TPPS = 0 correspond to those of the neat DHBC solutions at the same concentration as in the complexes (*C*DHBC = 0.2 mg/mL).

The observed gradual increase in *I*<sup>90</sup> values at both pH conditions serves as a proof of complexation and PIC formation, since the measured intensity is proportional to the mass of the scattering species in the solution. The apparent decrease at highest *C*TPPS and pH 3 is most probably an undesired effect of the filtration of the samples (a standard practice for DLS measurements so as to remove any large dust particles or aggregates), which is always associated with some degree of solute retention and of course is more pronounced at higher concentrations. At low *C*TPPS values (<0.06 mg/mL), the mass of the complexes formed at pH 3 is larger than the corresponding one at pH 7, a fact that indicates a higher degree of interaction and can be attributed to the increased charge density of the DHBC due to amine protonation. Nevertheless, as the concentration of the porphyrin increases (*C*TPPS > 0.07 mg/mL), an abrupt increase in the mass of the formed PICs at pH 7 is observed and likely denotes some degree of aggregation.

**Figure 1.** DLS intensity values at 90◦ *I*<sup>90</sup> for the DHBC/TPPS complex solutions at pH 7 and 3, as a function of porphyrin concentration *C*TPPS or charged groups ratio SO<sup>3</sup> −/NR<sup>2</sup> (R = CH<sup>3</sup> ).

Further insight regarding the properties of the complexes can be obtained from the equivalent size distribution functions (SDFs) extracted through the CONTIN regularization of the DLS measurements at 90◦ that are presented in Figure 2, along with the hydrodynamic radius *R*<sup>h</sup> values of the individual peaks as a function of *C*TPPS or the SO<sup>3</sup> <sup>−</sup>/NR<sup>2</sup> ratio at both pH values. The corresponding SDFs of the neat DHBC and TPPS solutions at relevant concentrations (*C*DHBC = 0.2 mg/mL and *C*TPPS = 0.053 mg/mL) are also included for comparison, with the extracted *R*<sup>h</sup> values being noted in Figure 2c as the point at *C*TPPS = 0 for the DHBC or the dashed line for TPPS.

With respect to the sizes of the species in solution, first of all we should point out that the DHBC exhibits two peaks, one small with an *R*<sup>h</sup> about 9 nm, which possibly represents the molecularly dissolved copolymer single chains, and a second larger one about 90 or 70 nm depending on the pH that most likely indicates some degree of self-assembly and the formation of multichain aggregates. Although both blocks of the PDMAEMA-*b*-POEGMA copolymer are deemed hydrophilic, hydrophobic interactions stemming from the hydrophobic nature of the polymeric backbone can never be excluded and thus lead to the proposed formation of multichain domains, as also previously observed in analogue systems [36,37]. However, the solutions of the complexes exhibit only one peak in all cases, suggesting the presence of monodisperse PICs with *R*<sup>h</sup> values ranging from 10 to 20 nm. As it seems upon interaction with the porphyrin the multichain domains of the DHBC breakup—likely because the electrostatic interactions in the system are stronger—and this way, only one type of complexes comprising of TPPS and single copolymer chains are formed. As the concentration of the porphyrin increases, so does the size of the PICs at both pH conditions, indicating further incorporation of TPPS. A more pronounced increase is evident at pH 3, leading to larger complexes, and is probably associated with the higher degree of interaction caused by protonation of the DHBC amine groups. Remarkably, the observed abrupt increase of the PICs mass at pH 7 and high *C*TPPS is not accompanied by a similar increase in size; therefore, it must be correlated with some type of conformational change that leads to more compact structures. One final observation here is that the neat porphyrin has a hydrodynamic radius of about 10 nm, surely significantly larger than its actual molecular size, which is about 20 Å [38]. Hence, we conclude that porphyrin is also aggregated to some extent.

**Figure 2.** Size distribution functions (SDFs) derived from DLS measurements at 90◦ for the DHBC/TPPS complex solutions at pH (**a**) 7 and (**b**) 3, along with (**c**) the *R*<sup>h</sup> values of the individual peaks as a function of *C*TPPS or SO<sup>3</sup> −/NR<sup>2</sup> ratio. The dashed line denotes the corresponding *R*<sup>h</sup> value of neat TPPS.

Another crucial characteristic of the formed PICs is their effective charge, which was acquired via electrophoretic light scattering (ELS) measurement, yielding the zeta potential *ζ*<sup>P</sup> values presented in an identical manner to the previous DLS results in Figure 3. At both pH values, a decrease in *ζ*<sup>P</sup> values of the complexes in regard to that of the neat DHBC (points at *C*TPPS = 0) is seen, suggesting the neutralization of charges that takes place upon interaction of the two oppositely charged species. At pH 7, we observe a broader change that eventually leads to charge inversion from positive to negative values, or in other words the negative charge of TPPS (*ζ*<sup>P</sup> ≈ −20 mV) prevails. On the contrary, at pH 3, though the initial decrease is more abrupt, the effective charge of the PICs remains positive for all complex solutions. This is apparently a consequence of the increased charge density of the positive DHBC (as also evident from the corresponding *ζ*<sup>P</sup> values) and the parallel reduction of TPPS negative charges (note that at pH 3 TPPS has a *ζ*<sup>P</sup> ≈ −10 mV) due to N protonation. Overall, the number of positive charges in the system is greater at pH 3, thus resulting in positively charged PICs.

**Figure 3.** Zeta potential *ζ*<sup>P</sup> values for the DHBC/TPPS complex solutions at pH 7 and 3, as a function of *C*TPPS or SO<sup>3</sup> −/NR<sup>2</sup> ratio. The dashed lines denote the corresponding *ζ*<sup>P</sup> values of neat TPPS.

Of equal importance to their solution characteristics are the optical properties of the formed PICs. For this reason, the complex solutions of the DHBC/TPPS system at both pH conditions were studied by UV-vis spectroscopy and the collected spectra are shown in Figure 4, where the corresponding spectra of the neat DHBC and TPPS solutions are also included for comparison. As expected, the DHBC does not exhibit any significant absorbance, so the observed peaks are attributed to the presence of the porphyrin. The spectroscopic features of TPPS are well known and can be directly correlated with the protonation and aggregation state of the porphyrin molecule [15–17,27,29,31,39]. As previously mentioned, at physiological pH values, TPPS exists in its free-base monomeric state (H2TPPS4−), which at high concentration or increased packing conditions can form face-to-face H-aggregates through *π*–*π* stacking of the molecules. On the other hand, at low pH values, the protonated diacid monomer (H4TPPS2−) is dominant and these zwitterionic molecules can interact electrostatically via their positive central and negative peripheral charges, forming side-by-side J-aggregates. Both types of TPPS monomers, as well as their aggregated counterparts, have distinctive absorption signatures.

In this case, TPPS solution at pH 7 shows a Soret band located at 413 nm accompanied by a Q-IV band at 516 nm, both indicative of the H2TPPS4<sup>−</sup> monomeric free-base form. In comparison, a gradual blue shift of about 8 nm is observed for the Soret band of the complex solutions, peaking at around 405 nm, while at the same time the Q-bands are slightly shifted to higher wavelengths (red shift). These two findings are related to the formation of H-type aggregates [15–17], an apparent consequence of the complexation with the DHBC, which increases the proximity of the porphyrin molecules and thus enhances their aggregation. Moreover, it is worth noting that for the complex solution with the highest *C*TPPS, the Soret band shows also significant contribution from the 413 nm peak. This probably signifies the coexistence of H-aggregates and free-base monomers, meaning that some uncomplexed porphyrin molecules are still present in the solution. Therefore, it is possible that the maximum binding capacity of the DHBC has been exceeded.

**Figure 4.** UV-vis spectra of the DHBC/TPPS complex solutions at pH (**a**) 7 and (**b**) 3. The insets show a scaling up of the Q-bands area. The corresponding spectra of neat TPPS and DHBC are also included for comparison.

At pH 3, the neat porphyrin shows a Soret band again peaking at 413 nm—the spectroscopic signature of the free-base monomer—but also a shoulder at 432 nm is observed. This shoulder, along with the distinctive Q-I peak at 644 nm, denote the presence of the diacid monomer [15–17]. Interestingly, it seems that both types of monomeric forms (H2TPPS4<sup>−</sup> and H4TPPS2−) are simultaneously present in the solution. A possible explanation for this is that the aggregation of the porphyrin molecules hinders their protonation to a full extent. It should be mentioned here that although no signs of H-aggregates can be seen in the spectra of neat TPPS at pH 7, these measurements were performed on highly diluted solutions (1:100) so as not to exceed maximum allowed absorbance values. Surely, the presence of aggregates in the initial stock solutions (*C*TPPS = 0.53 mg/mL) that were used for the preparation of the PICs cannot be excluded, as was also evidenced by the corresponding hydrodynamic radii (*R*<sup>h</sup> ≈ 10 nm). In regard to the complexes, the Soret band is once more shifted at 405 nm and the Q-bands are slightly red-shifted, denoting the existence of H-aggregates. Nevertheless, as porphyrin concentration increases the representative peaks of J-aggregates at 490 and 701 nm appear [15–17], as clearly obvious in the case of Comp (2 + 2) but also noticeable for the solutions of Comp (2 + 1.4) and neat TPPS, as seen in the inset of Figure 4b. Apparently, at higher *C*TPPS values the number of TPPS molecules participating in the complexes and hence their proximity increases drastically, leading to J-type aggregation. Lastly, no signs of uncomplexed TPPS can be discerned in this pH value, one more indication of the increased interaction capability of the DHBC due to the protonation of its amine groups.

Fluorescence spectroscopy measurements of the same PIC solutions were also performed and the relative emission spectra are shown in Figure 5. Overall, the fluorescence spectral features corroborate the findings from the UV-vis measurements. At pH 7, the neat TPPS solution displays a double peak at 641 and 698 nm, which is typical of the free-base monomer [16,27,29]. A red shift of about 20 nm is observed for the complex solutions with the main emission peak appearing at 663 nm, thus signifying the presence of H-aggregates. Notably, as porphyrin concentration increases, contribution from the 641 nm peak becomes more and more prominent, culminating in the case of the Comp (2 + 2) solution, again indicating uncomplexed TPPS monomeric molecules. Respectively, at pH 3 a significant decrease in the fluorescence intensity of the neat TPPS is observed and the main peak is located around 677 nm, both spectral traits of the diacid H4TPPS2<sup>−</sup> form [16,27,29]. However, a small shoulder at 636 nm probably correlated with the free-base monomer is still discerned. The obtained emission spectra for the solutions of the PICs resemble the ones at pH 7, with the main peak about 667 nm (existence of H-aggregates). For the two complex solutions with higher porphyrin concentration, extended quenching is observed along with

the appearance of the peak at 732 nm, transitions that can be attributed to the formation of J-aggregates [16,27,29].

**Figure 5.** Fluorescence spectra of the DHBC/TPPS complex solutions at pH (**a**) 7 and (**b**) 3. The corresponding spectra of neat TPPS and DHBC are also included for comparison.

#### *3.2. QPDMAEMA-b-POEGMA and TPPS complexation*

Undoubtedly, electrostatic interactions play the most crucial role during the coupling of (macro)molecular species bearing opposite charges and essentially govern not only the complexation process itself but the properties of the resulting nanoparticles as well. Ergo, any change in the charged state of the individual components is expected to influence the final outcome and for this reason is worth investigating. On these grounds, we also studied the interaction of the quaternized homologue QPDMAEMA-*b*-POEGMA copolymer, which is a strong polyelectrolyte independently of pH with the TPPS porphyrin at both acidic and neutral pH conditions. Figure 6 presents the collective DLS and ELS results attained for the QDHBC/TPPS system in regard to scattering intensity, hydrodynamic radius, size distribution and effective charge, as a function of *C*TPPS or equivalently the SO<sup>3</sup> <sup>−</sup>/NR<sup>3</sup> + (R = CH3) charged groups ratio at both pH 7 and 3.

As seen in Figure 6a, the mass of the complexes (proportional to the scattering intensity) increases with increasing *C*TPPS, validating once again their formation. Somewhat larger *I*<sup>90</sup> values are observed at pH 3 than at 7, although in this case the charge density of the QDHBC is the same at both pH values. Possibly, the higher mass of the complexes at pH 3 could be attributed to the differences in the protonation and/or aggregation state of the porphyrin molecule, as well as hydration state of the copolymer due to the additional CH<sup>3</sup> group. Moreover, generally higher scattering values are recorded in comparison to the previous DHBC/TPPS system (see Figure S1), indicating an analogous difference in the mass of the corresponding PICs. Quite interestingly, abrupt increase in *I*<sup>90</sup> at pH 7 occurs only at the highest porphyrin concentration, demonstrating a more gradual and less intense transition to more compact conformations.

A better understanding of the observed changes in the mass of the complexes can be gained by examining the corresponding differences in their size and size distributions (Figure 6b–d). First of all, the QDHBC shows two peaks signifying the presence of single copolymer chains with a size of about 5 nm, along with multichain domains/aggregates giving peaks about 70 or 80 nm (at pH 7 or 3, respectively), as was the case for the precursor copolymer. The remarkable difference for this system is that the majority of the complex solutions also exhibit two peaks, which most likely denote the presence of two different types of complexes in regard to the morphology of the QDHBC. In other words, both the single chains and the multichain aggregates of the copolymer form complexes with the porphyrin. At pH 7 and high *C*TPPS, the second peak is no longer discerned, suggesting that the multichain aggregates break up as the interaction with the TPPS becomes more prominent and only one type of PIC is preserved. On the contrary, at pH 3

both types of PICs coexist throughout the whole range of mixing ratios (i.e., component analogies), a fact that clearly justifies the observed increased mass. As it seems the different protonation/aggregation state of TPPS at pH 3 entails a different type of interaction with the quaternized copolymer, presumably weaker, that apparently is not able to cause the dissociation of the multichain domains. A small increase in most peaks (apart from *R*h2 at pH 7) can be seen, possibly indicating the incorporation of more porphyrin molecules.

**Figure 6.** Collective DLS and ELS results in regard to (**a**) the scattering intensity at 90◦ , (**b**) the hydrodynamic radius derived from (**c**,**d**) the corresponding size distribution functions (SDFs) at 90◦ , and (**e**) zeta potential values for the QDHBC/TPPS complex solutions at pH 7 and 3, as a function of porphyrin concentration *C*TPPS or charged groups ratio SO<sup>3</sup> −/NR<sup>3</sup> + (R = CH<sup>3</sup> ).

As far as the effective charge of the complexes is concerned, the *ζ*<sup>P</sup> values decrease as *C*TPPS increases (Figure 6e), a direct and anticipated consequence of the charge neutralization that takes place upon interaction. Again, the overall change is greater at pH 7 than

at 3 owing to the increased number of positive charges related to TPPS protonation and aggregation state.

Additional information about the state of the porphyrin molecules in the complexes can be derived from the UV-vis and fluorescence spectra of the QDHBC/TPPS system at both pH values shown in Figure 7. Absorbance spectra at pH 7 are almost identical to the ones measured for the previous system, characterized by a blue-shifted Soret band peaking about 404 nm and somewhat red-shifted Q-bands indicative of H-aggregation. Similarly, the same observations regarding the formation of H-type aggregates can be made at pH 3, while at high porphyrin concentrations the distinctive spectral features of J-aggregates are ascertained (peaks at 491 and 703 nm). Note that at both pH conditions and highest porphyrin content, the contribution of the 413 nm peak is clearly evident in the Soret band of the complexes, revealing the existence of uncomplexed TPPS and thus implying that the maximum binding capacity of the quaternized copolymer has been reached. Fluorescence spectroscopy measurements confirm the attained rationalization about H-aggregation by showing at both pH 7 and 3 a main peak located around 667 nm. Nevertheless, a more pronounced quenching of the emission signal occurs in this case, meaning that the aggregation of the porphyrin molecules is more extensive. Furthermore, at pH 3 and high *C*TPPS, telltale signs of J-aggregates (extensive quenching and peak at 732 nm) can be seen.

**Figure 7.** UV-vis (**a**,**b**) and fluorescence (**c**,**d**) spectra of the QDHBC/TPPS complex solutions at pH 7 (left) and 3 (right). The insets in the top row show a scaling up of the Q-bands area. The corresponding spectra of neat TPPS and QDHBC are also included for comparison.

#### *3.3. Comparison of the Two Systems in Different Conditions*

Certainly, the sum of the results presented thus far emphasizes the importance of self-assembly processes governed by electrostatic and/or hydrophobic interactions when one deals with such nanostructured hybrid systems. According to our observations, the nature of the macromolecular copolymer in regard to its protonation degree or equivalent charge density, as well as solvation state and latent hydrophobic nature that leads to the formation of multichain domains, along with the complementary features of the porphyrin molecule mainly involving its protonation and aggregation state, constitute the key factors influencing the structure and properties of the resulting PICs. Recapitulating, both the precursor PDMAEMA-*b*-POEGMA copolymer and its quaternized counterpart form multichain aggregates at either pH 7 or 3 (apparently stemming from hydrophobic backbone interactions) coexisting with single polymeric chains in solution. In an analogue manner, TPPS porphyrin exhibits two protonation states, that is, the free-base (H2TPPS4−) at pH 7 or diacid (H4TPPS2−) at pH 3 monomer, which in turn self-assemble when in close proximity into H- or J-type structures due to *π*–*π* stacking or electrostatic binding, respectively. Upon mixing of these different species in the case of the DHBC/TPPS system at low porphyrin concentration and both pH conditions, monodisperse PICs comprising H-aggregated TPPS molecules and singe DHBC chains are formed. As the porphyrin content increases, so does its aggregation, leading to more compact complexed H-aggregates at pH 7, integrating more of TPPS molecules and eventually exceeding DHBC binding capacity. Accordingly, at pH 3 both H- and J-aggregates are incorporated in the resulting complexes, since both types are present in the corresponding neat TPPS solution, while the increased charge density of the DHBC causes a higher degree of interaction (no uncomplexed TPPS is discerned). When it comes to the quaternized DHBC, it seems that the consecutive change in the hydration state of the copolymer is associated with more stable multichain aggregates that do not easily dissociate during complexation. This way, two types of distinct PICs are formed at both pH values and low *C*TPPS corresponding to single and multichain copolymer–porphyrin complexes. However, as the porphyrin concentration becomes higher at pH 7 only the single-chain type of PICs remains incorporating more TPPS molecules (suggesting that porphyrin aggregation is more prominent), while at pH 3 both types coincide and even complexes of J-aggregated TPPS are observed. Still, at both pH conditions spectroscopic signs of uncomplexed porphyrin are recorded, a fact that implies a reduced binding affinity for the quaternized copolymer in comparison to the precursor DHBC, a counterintuitive finding that points out the significance of the intrinsic polymeric solution properties. This proposed interaction scenario is schematically depicted in the following illustration (Scheme 2).

#### *3.4. Temperature Effect on the Formed PICs*

The ability of photosensitizers intended for clinical applications to respond to externally applied stimuli such as temperature is of particular interest, since it can open new treatment pathways and even enable combination of therapeutic protocols or methods, thus enhancing their efficiency potential [4,40]. To this end, we investigated the effect of temperature on the already formed PICs of both systems under study. Figure 8 shows the summary of DLS results regarding the values of the scattering intensity *I*<sup>90</sup> and the hydrodynamic radius *R*<sup>h</sup> derived from the corresponding size distribution functions (SDFs) presented in Figures S2–S4 obtained via measurements performed at 90◦ and different temperatures ranging from 25 to 60 °C (with a 5 °C increment), for two representative solutions of complexes-Comp (2 + 1) and Comp (2 + 2)-of the DHBC or QDHBC/TPPS system at both pH 7 and 3. The corresponding values of the neat copolymers and porphyrin solutions are also included for comparison. In a similar manner, fluorescence spectra of the same representative solutions measured at temperatures of 25, 40, 60 °C, and back at 25 °C after heat treatment are displayed in Figure 9, along with the corresponding spectra of neat TPPS.

**Scheme 2.** Proposed interaction illustration for the formation of PICs between the DHBC or its quaternized counterpart QDHBC and TPPS porphyrin at different pH values and porphyrin contents.

At first, the effect of temperature on the pure constituents of the complex systems is presented. For both block copolymers, their mass (proportional to *I*90) seems rather constant throughout the range of studied temperatures. In regard to their size, although the *R*<sup>h</sup> of the first peak (single copolymer chains) shows small changes, an apparent decrease in the *R*<sup>h</sup> of the second peak is observed (see also Figure S4). This fact indicates that the multichain aggregates shrink as temperature increases, most probably because hydrophobic interactions become stronger. When temperature drops back to ambient conditions, all sizes are restored. As far as porphyrin is concerned, its size is almost unaffected but a systematic decrease of its mass can be discerned, especially in the case of pH 3, where it is more pronounced. This decrease of mass is also accompanied by a significant transition in the respective fluorescence spectra, with the one at 60 °C showing clear signs of the free-base monomer (Figure 9e). Apparently, disaggregation (at least to some extent) of the porphyrin H-aggregates-present at both pH values, as previously discussed-takes place, which is eventually reestablished when the solution cools down.

For the complex solutions of the DHBC/TPPS system, only small changes in their mass are observed, apart from the noticeable decrease of the scattering intensity in the case of the Comp (2 + 2) solution at pH 7, which in combination with the parallel increase in emission intensity could denote a dissociation of the aggregated porphyrin molecules. At the same time, their sizes are either stable or suggest slight shrinking as a consequence of enhanced hydrophobic interactions. This seems to also be the case for the smaller population of complexes of the QDHBC/TPPS system, the one that corresponds to single copolymer chain–porphyrin complexes. Remarkably, the larger complexes that are correlated with the copolymer multichain aggregates either diminish or even disappear completely upon heating. This change is also expressed as an increase in the fluorescence intensity or in other words dequenching, denoting possible disaggregation of TPPS. Most likely at higher temperatures, the interaction between the copolymer and porphyrin molecules is more favorable, facilitated also by the apparent dissociation of porphyrin aggregates, thus leading to the dissociation of the multichain complexes. Overall, the increase in temperature showed that the formed PICs are susceptible to conformational rearrangements so as to compensate for the loss of hydration due to the stronger hydrophobic interactions.

**Figure 8.** DLS results in regard to (**a**,**c**) the scattering intensity at 90◦ and (**b**,**d**) the hydrodynamic radius derived from the corresponding size distribution functions (SDFs) at 90◦ , for the complex solutions (Q)Comp (2 + 1) and (Q)Comp (2 + 2) of the (Q)DHBC/TPPS systems at pH 7 (left) and 3 (right) at different temperatures ranging from 25 to 60 °C (5 °C step), and cooled back at 25 °C after heat treatment (AHT). The corresponding values of neat (Q)DHBC and TPPS are also included for comparison.

**Figure 9.** Fluorescence spectra of the complex solutions (**a**,**c**) (Q)Comp (2 + 1) and (**b**,**d**) (Q)Comp (2 + 2) of the (Q)DHBC/TPPS systems at pH 7 (left) and 3 (right) at different temperatures: 25, 40, 60 °C, and 25 °C after heat treatment (AHT). (**e**) The corresponding spectra of neat TPPS for comparison.

#### **4. Conclusions**

The electrostatic complexation between the DHBC PDMAEMA-*b*-POEGMA and the corresponding quaternized strong block polyelectrolyte QPDMAEMA-*b*-POEGMA with TPPS porphyrin led to the formation of nanostructured photoactive PICs. Both the solution and optical properties and the morphology and structure of these complexed hybrid species proved to be dependent on the protonation and aggregation state of their original constituents, the pH of the solution, and the analogy between the two components. Monodisperse small PICs were formed in the case of the DHBC/TPPS system at both pH 7 and 3, consisting of single copolymer chains and H- or J-type aggregates of TPPS depending on the pH. The increase in TPPS content resulted in more compact complexed porphyrin aggregates. Respectively, the analogous QDHBC/TPPS system was characterized by the presence of two different types of complexes, those resulting from the interaction of the single chains or the multichain aggregates of the quaternized copolymer and the corresponding H-aggregates of TPPS at both pH values and low porphyrin contents. At higher TPPS concentration, increased aggregation of the porphyrin occurred, subsequently affecting the structure of the complexes and thus leading to larger H- or even J-type complexed aggregates. The optical properties of the PICs for both systems were directly correlated with the intrinsic protonation and/or aggregation state of TPPS, which was evidently further enhanced upon interaction with the copolymers. Actually, the quaternized counterpart caused a greater degree of porphyrin aggregation compared to the precursor DHBC. Finally, the formed PICs responded to the increase in temperature by appropriate conformational rearrangements that led to disaggregation of the complexed porphyrin in the case of multichain–porphyrin complexes or more compact structures (shrinking) for the single-chain ones.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/polym14235186/s1, Figure S1: Comparison of DLS scattering intensity values at 90◦ *I*<sup>90</sup> for the DHBC/TPPS (closed symbols) and the QDHBC/TPPS (open symbols) complex solutions at pH 7 and 3, as a function of porphyrin concentration *C*TPPS or charged groups ratio SO<sup>3</sup> −/NR<sup>2</sup> or NR<sup>3</sup> + (R = CH<sup>3</sup> ).; Figure S2: Size distribution functions (SDFs) derived from DLS measurement at 90◦ for the complex solutions (a, b) Comp (2 + 1) and (c, d) Comp (2 + 2) of the DHBC/TPPS system at pH 7 (left) and 3 (right) at different temperatures ranging from 25 to 60 °C (5 °C step), and cooled back to 25 °C after heat treatment (AHT); Figure S3: Size distribution functions (SDFs) derived from DLS measurement at 90◦ for the complex solutions (a,b) QComp (2 + 1) and (c, d) QComp (2 + 2) of the QDHBC/TPPS system at pH 7 (left) and 3 (right) at different temperatures ranging from 25 to 60 °C (5 °C step), and cooled back to 25 °C after heat treatment (AHT); Figure S4: Size distribution functions (SDFs) derived from DLS measurement at 90◦ for the neat (a,b) DHBC, (c,d) QDHBC and (e,f) TPPS solutions at pH 7 (left) and 3 (right) at different temperatures ranging from 25 to 60 °C (5 °C step), and cooled back to 25 °C after heat treatment (AHT).

**Author Contributions:** Conceptualization, S.P.; methodology, D.K and M.K.; formal analysis, D.K. and M.K.; data acquisition, D.K.; data curation, D.K. and M.K.; writing—original draft preparation, M.K.; writing—review and editing, S.P.; supervision, M.K. and S.P. 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:** Data presented in this study are available on reasonable request from the corresponding author.

**Acknowledgments:** The authors would like to express their gratitude to Varvara Chrysostomou for the synthesis of the copolymers used in this study.

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

### **References**

