Physicochemical Evaluation of Insulin Complexes with QPDMAEMA-b-PLMA-b-POEGMA Cationic Amphiphlic Triblock Terpolymer Micelles

Herein, poly[quaternized 2-(dimethylamino)ethyl methacrylate-b-lauryl methacrylate-b-(oligo ethylene glycol)methacrylate] (QPDMAEMA-b-PLMA-b-POEGMA) cationic amphiphilic triblock terpolymers were used as vehicles for the complexation/encapsulation of insulin (INS). The terpolymers self-assemble in spherical micelles with PLMA cores and mixed QPDMAEMA/POEGMA coronas in aqueous solutions. The cationic micelles were complexed via electrostatic interactions with INS, which contains anionic charges at pH 7. The solutions were colloidally stable in all INS ratios used. Light-scattering techniques were used for investigation of the complexation ability and the size and surface charge of the terpolymer/INS complexes. The results showed that the size of the complexes increases as INS ratio increases, while at the same time the surface charge remains positive, indicating the formation of clusters of micelles/INS complexes in the solution. Fluorescence spectroscopy measurements revealed that the conformation of the protein is not affected after the complexation with the terpolymer micellar aggregates. It was observed that as the solution ionic strength increases, the size of the QPDMAEMA-b-PLMA-b-POEGMA/INS complexes initially decreases and then remains practically constant at higher ionic strength, indicating further aggregation of the complexes. atomic force microscopy (AFM) measurements showed the existence of both clusters and isolated nanoparticulate terpolymer/protein complexes.


Introduction
Diabetes mellitus is one of the most common and serious chronic diseases, affecting millions of patients worldwide [1,2]. The discovery of insulin was one of the most important scientific achievements of the last century [3]. Patients suffering from diabetes need to be administered with several doses of insulin daily, to maintain their blood glycose levels in the desirable rates. The main way for insulin administration is through subcutaneous injections [4].
Therefore, the need exists for the development of novel, effective nanocarriers that will employ alternative routes, such as oral administration, for the delivery of insulin. Presently, the evolution of synthetic polymer chemistry gives scientists the ability to design and synthesize polymers with tailored properties, a very important feature when it comes to gene/protein delivery applications [5,6]. Block polyelectrolytes are a very attractive class of polymers and they have been effectively used as nanocarriers for the delivery of genes and proteins, since they offer significant advantages, such as small size, good solubility and colloidal stability in aqueous solutions and high cellular uptake efficiency [7][8][9][10][11][12]. The complexation between polyelectrolytes and genes/proteins is mainly achieved via electrostatic interactions [13][14][15][16].
Cationic block polyelectrolytes have been widely used for the delivery of DNA [9,17] and proteins [18,19] since they carry positive charges that interact with the oppositely charged genes/proteins. Block polyelectrolyte design plays an important role in the structure and physicochemical/biological properties of the complexes formed.
Polyelectrolytes offer certain advantages over other nanocarriers when it comes to insulin delivery applications, such as nanoscale size, protection from degradation and controlled release [2,18,20,21]. Moreover, such nanocarriers facilitate the uptake of insulin from routes other than invasive [2,4].
In this work, QPDMAEMA-b-PLMA-b-POEGMA cationic triblock terpolymer micelles were used as nanocarriers for insulin (INS). The formation of the terpolymer/INS complexes was achieved through electrostatic interactions between the positive charges of the micelles and the negative charges on insulin. The complexation process was investigated, in a physicochemical aspect, by dynamic and electrophoretic light-scattering (DLS, ELS), atomic force microscopy (AFM) and fluorescence spectroscopy (FS).
Polymers 2020, 12, x FOR PEER REVIEW 2 of 12 used as nanocarriers for the delivery of genes and proteins, since they offer significant advantages, such as small size, good solubility and colloidal stability in aqueous solutions and high cellular uptake efficiency [7][8][9][10][11][12]. The complexation between polyelectrolytes and genes/proteins is mainly achieved via electrostatic interactions [13][14][15][16]. Cationic block polyelectrolytes have been widely used for the delivery of DNA [9,17] and proteins [18,19] since they carry positive charges that interact with the oppositely charged genes/proteins. Block polyelectrolyte design plays an important role in the structure and physicochemical/biological properties of the complexes formed.
Polyelectrolytes offer certain advantages over other nanocarriers when it comes to insulin delivery applications, such as nanoscale size, protection from degradation and controlled release [2,18,20,21]. Moreover, such nanocarriers facilitate the uptake of insulin from routes other than invasive [2,4].
In this work, QPDMAEMA-b-PLMA-b-POEGMA cationic triblock terpolymer micelles were used as nanocarriers for insulin (INS). The formation of the terpolymer/INS complexes was achieved through electrostatic interactions between the positive charges of the micelles and the negative charges on insulin. The complexation process was investigated, in a physicochemical aspect, by dynamic and electrophoretic light-scattering (DLS, ELS), atomic force microscopy (AFM) and fluorescence spectroscopy (FS).

Methods
Dynamic light-scattering measurements were conducted on an ALV/CGS-3 compact goniometer system (ALVGmbH, Hessen, Germany), equipped with an ALV 5000/EPP multi-τ digital correlator with 288 channels and an ALV/LSE-5003 light-scattering electronics unit for stepper motor drive and limit switch control. A JDS Uniphase 22 mW He-Ne laser (λ = 632.8 nm) was used as the light source. Measurements of the intensity correlation function were carried out five times for each concentration and angle and were averaged for each angle. The solutions were filtered through 0.45 µm hydrophilic PTFE filters (Millex-LCR from Millipore, Billerica, MA, USA) before measurements. The angular range for the measurements was 30-150 • . Obtained correlation functions were analyzed by the cumulants method and the CONTIN software (ALVGmbH, Hessen, Germany). The size data and figures shown below are from measurements at 90 • .
For the ionic strength dependent light-scattering measurements, the ionic strength of the polymer/INS solution increased by gradual addition of the appropriate volume of NaCl (using a 1 M stock solution). After each addition, the solution was rigorously stirred and left to equilibrate for 15 min before measurement.
AFM measurements were performed in the semicontact (tapping) mode under ambient conditions using a NT-MDT NTEGRA Prima scanning probe microscope (NT-MDT Spectrum Instruments, Moscow, Russia) equipped with a Nanosensors silicon cantilever (Nanosensors, Neuchâtel, Switzerland). Aqueous polymeric solution (ca. 5 × 10 −4 g/mL) were deposited on a freshly peeled out mica surface (flogopite, Geological Collection of Charles University in Prague, Czech Republic). The samples were dried in a vacuum oven at ambient temperature for 24 h.
Picosecond time-resolved fluorescence spectra were measured by the time-correlated single photon counting (TCSPC) method on a Nano-Log spectrofluorometer (Horiba JobinYvon, Kyoto, Japan), by using a laser diode as an excitation source (NanoLED, 375 nm) and a UV−vis detector TBX-PMT series (250−850 nm) by Horiba JobinYvon. Lifetimes were evaluated with the DAS6 Fluorescence-Decay Analysis Software (Kyoto, Japan).

Results and Discussion
The ability of QPDMAEMA-b-PLMA-b-POEGMA cationic triblock terpolymer micelles to complex with insulin through electrostatic interactions is investigated. The terpolymer/protein complexes were prepared in various INS concentrations, within the range C INS = 0.125−0.5 mg·mL −1 . The molecular characteristics of the terpolymers used are presented in Table 1. A schematic illustration of the formation of complexes from QPDMAEMA-b-PLMA-b-POEGMA triblock terpolymer micelles and insulin is presented in Scheme 2. Protein globules are expected to be complexed with the QPDMAEMA chains of the micellar corona and to occupy all available space within the corona. Some protein molecules located close to the periphery of the micelles act as bridges and enhance the formation of clusters of complexes. The neutral hydrophilic POEGMA chains in the corona contribute to the colloidal stabilization of the complexes/clusters. Size and surface charge are very important parameters for the determination of the efficiency of polymeric systems in protein delivery applications. Thus, the complexes of QPDMAEMA-b-PLMA-b-POEGMA triblock terpolymers with insulin were investigated by light-scattering techniques (dynamic and electrophoretic) to gain information about their size and surface charge, respectively. It must be mentioned that all terpolymer/INS solutions prepared were colloidally stable and no precipitation phenomena were observed. This is a very important observation, especially for the complexes formed with QPDMAEMA13-b-PLMA39-b-POEGMA8 triblock terpolymer, which has the highest hydrophobic block (PLMA) ratio. Size and surface charge are very important parameters for the determination of the efficiency of polymeric systems in protein delivery applications.
Thus, the complexes of QPDMAEMA-b-PLMA-b-POEGMA triblock terpolymers with insulin were investigated by light-scattering techniques (dynamic and electrophoretic) to gain information about their size and surface charge, respectively. It must be mentioned that all terpolymer/INS solutions prepared were colloidally stable and no precipitation phenomena were observed. This is a very important observation, especially for the complexes formed with QPDMAEMA 13 -b-PLMA 39 -b-POEGMA 8 triblock terpolymer, which has the highest hydrophobic block (PLMA) ratio. Figure 1a depicts size distribution graphs from DLS measurements (by CONTIN analysis) for QPDMAEMA 13  It was observed that both the scattering intensity and R h of the complexes increase as INS concentration increases, for both terpolymers used (Figure 2a R h value is~80 nm. Additionally, the fact that the size of the particles is larger than the size of the polymeric micelles before the complexation with insulin is a proof for the successful formation of the terpolymer/INS complexes. It was observed that both the scattering intensity and Rh of the complexes increase as INS concentration increases, for both terpolymers used (Figure 2a   The surface charge of all terpolymer/INS complexes solutions prepared was also investigated. It can be seen that the zeta potential (ζp) values for QPDMAEMA33-b-PLMA16-b-POEGMA30/INS complexes are positive (Figure 2c), a result that confirms the scenario about the formation of clusters of complexes, as discussed earlier, which also implies that the periphery of the complexes (or clusters of complexes) are populated by terpolymer micelles and in particular cationic segments, mostly hiding the protein molecules in the interior of the clusters. For this to happen, each molecule of insulin must interact with more than one polymeric micelle forming bridges between terpolymer micelles. Furthermore, the ζp values become more positive (without large alterations) as INS concentration increases, indicating that the highest the INS concentration the highest the tendency and by extension, the lowest number of positive charges in the micelles of this particular terpolymer, which facilitates the formation of more compact clusters of complexes compared to the previous case.
The surface charge of all terpolymer/INS complexes solutions prepared was also investigated. It can be seen that the zeta potential (ζ p ) values for QPDMAEMA 33 -b-PLMA 16 -b-POEGMA 30 /INS complexes are positive (Figure 2c), a result that confirms the scenario about the formation of clusters of complexes, as discussed earlier, which also implies that the periphery of the complexes (or clusters of complexes) are populated by terpolymer micelles and in particular cationic segments, mostly hiding the protein molecules in the interior of the clusters. For this to happen, each molecule of insulin must interact with more than one polymeric micelle forming bridges between terpolymer micelles. Furthermore, the ζ p values become more positive (without large alterations) as INS concentration increases, indicating that the highest the INS concentration the highest the tendency for the formation of clusters of complexes.
In the case of QPDMAEMA 13 -b-PLMA 39 -b-POEGMA 8 /INS complexes, ζ p values are more positive and show a small decrease as INS concentration in the solution increases (Figure 2d). This probably means that a higher number of cationic segments populate the periphery of the clusters in this case and there are some subtle differences in the morphology of the clusters compared to the previous case. This behavior may be due to the QPDMAEMA block length being shorter in the QPDMAEMA 13 -b-PLMA 39 -b-POEGMA 8 terpolymer.
According to literature, an increase in the ionic strength of the solutions of polyelectrolyte complexes with peptides or proteins can lead either to complex dissociation, since the electrostatic interactions between the components become weaker as a result of screening effects, or to secondary aggregation, or to precipitation of the original complexes due to lowering of solvent quality for the dispersed particles [26][27][28]. Figure 3 shows the variations in the scattering intensity and hydrodynamic radius, as a function of ionic strength, for the QPDMAEMA 33 -b-PLMA 16  The scattering intensity and hydrodynamic radius of QPDMAEMA13-b-PLMA39-b-POEGMA8/INS complexes as a function of ionic strength, present similar behavior with the one discussed above at the corresponding insulin concentrations. Therefore, as far as the solution ionic strength effects on the structure and stability are concerned these are more pronounced and distinct for the two terpolymer/insulin systems at lower concentrations of insulin.    Figure  4. The existence of both isolated particles (primary micelle/protein complexes) and aggregates (clusters of primary complexes) with average size in the range of 130-150 nm (diameter) is observed, as well as some larger aggregates formed by coalescence of the species on the mica surface. The height of the particles has been found to be around 100 nm, smaller than the dimensions at xy level, meaning that there is interaction with the substrate (mica) or that the structure of the complexes is rather loose and collapse of the structures occurs after their deposition on the substrate and the solvent removal. Such a loose structure is expected for polyelectrolyte/protein complexes due to their hydrophilic character and their ability to trap water in their interior. Even in the case of terpolymer micelles the larger part of the polymeric component is taken up by the swollen hydrophilic mixed QPDMAEMA/POEGMA corona compared to the space occupied by the hydrophobic PLMA cores. The scattering intensity and hydrodynamic radius of QPDMAEMA 13 -b-PLMA 39 -b-POEGMA 8 /INS complexes as a function of ionic strength, present similar behavior with the one discussed above at the corresponding insulin concentrations. Therefore, as far as the solution ionic strength effects on the structure and stability are concerned these are more pronounced and distinct for the two terpolymer/insulin systems at lower concentrations of insulin.
AFM measurements were performed to have a more complete picture about the morphology of QPDMAEMA-b-PLMA-b-POEGMA/INS complexes. Indicative AFM images are presented in Figure 4. The existence of both isolated particles (primary micelle/protein complexes) and aggregates (clusters of primary complexes) with average size in the range of 130-150 nm (diameter) is observed, as well as Polymers 2020, 12, 309 8 of 11 some larger aggregates formed by coalescence of the species on the mica surface. The height of the particles has been found to be around 100 nm, smaller than the dimensions at xy level, meaning that there is interaction with the substrate (mica) or that the structure of the complexes is rather loose and collapse of the structures occurs after their deposition on the substrate and the solvent removal. Such a loose structure is expected for polyelectrolyte/protein complexes due to their hydrophilic character and their ability to trap water in their interior. Even in the case of terpolymer micelles the larger part of the polymeric component is taken up by the swollen hydrophilic mixed QPDMAEMA/POEGMA corona compared to the space occupied by the hydrophobic PLMA cores.     Figure 5a and the fluorescence intensity at peak maximum graphs as a function of INS concentration at 300 nm (peak maximum wavelength) are presented in Figure 5b. The deviation of the wavelength in which the maximum protein fluorescence intensity is observed is less than 10 nm, showing that there are no significant changes in the conformation of the protein after the formation of terpolymer/INS complexes. It is obvious that the fluorescence intensity increases as INS concentration in the solution increases in a rather linear fashion showing no precipitation in the solutions of the complexes (and in some way the stability of the complexes as insulin concentration increases).
Time-resolved FS measurements were performed on QPDMAEMA-b-PLMA-b-POEGMA/INS complexes to investigate the events that take place during the lifetime of the excited singlet state of the intrinsic tyrosine fluorescence. The results are presented in Figure 6. An increase in the relaxation time (average values shown in Figure 6) is observed as INS concentration in the solution increases. The increase is dramatic if compared with the relaxation time for free INS. This observation can be attributed to a more stereochemically constrained environment for tyrosine, and subsequently for significant crowding of the whole protein molecules participating in the complexes, because of the strong complexation with the cationic QPDMAEMA chains and their localization within the palisade of the micellar corona.

Conclusions
The ability of QPDMAEMA-b-PLMA-b-POEGMA cationic triblock terpolymer micelles to form complexes with insulin was demonstrated in this work. The positively charged micelles interact electrostatically with the negatively charged insulin forming complexes and clusters of complexes.
All the QPDMAEMA-b-PLMA-b-POEGMA/INS complexes solutions that were colloidally stable at all insulin concentrations used. The size of the complexes/clusters was found to increase as the protein concentration in the solution increased and the positive values of ζ-potential shows that the addition of insulin leads to formation of aggregates with a large number of positively charged segments in the periphery. This observation is also supported from AFM measurements, showing the existence of aggregates and single particles (complexes) in the solution. FS measurements show that the conformation of the protein is not affected after the complexation with the polymeric micelles and that the protein experiences a largely constrained environment within the complexes/clusters. Thus, the terpolymers can be used further for insulin delivery applications, due also to the small size of QPDMAEMA-b-PLMA-b-POEGMA/INS complexes formed.

Conclusions
The ability of QPDMAEMA-b-PLMA-b-POEGMA cationic triblock terpolymer micelles to form complexes with insulin was demonstrated in this work. The positively charged micelles interact electrostatically with the negatively charged insulin forming complexes and clusters of complexes.
All the QPDMAEMA-b-PLMA-b-POEGMA/INS complexes solutions that were colloidally stable at all insulin concentrations used. The size of the complexes/clusters was found to increase as the protein concentration in the solution increased and the positive values of ζ-potential shows that the addition of insulin leads to formation of aggregates with a large number of positively charged segments in the periphery. This observation is also supported from AFM measurements, showing the existence of aggregates and single particles (complexes) in the solution. FS measurements show that the conformation of the protein is not affected after the complexation with the polymeric micelles and that the protein experiences a largely constrained environment within the complexes/clusters. Thus, the terpolymers can be used further for insulin delivery applications, due also to the small size of QPDMAEMA-b-PLMA-b-POEGMA/INS complexes formed.