*2.3. Synthesis of the Hybrid-Polypeptides*

The synthesized polymers are illustrated in Scheme 1. The synthetic procedure is described in detail in Supplementary Materials. Briefly, the synthesis of the hybrid polypeptide terpolymers was achieved through a ring-opening polymerization (ROP) process [39–41] of the corresponding *N*-carboxy anhydrides, using an amino end-functionalized poly(ethylene oxide) (*m*-PEO-NH2) macroinitiator, with molecular weight 10.0 × 103 g mol<sup>−</sup>1. Highly purified DMF was the solvent at all polymerizations. In case of the *m*PEO-*b*-PHis-*b*-PCys as well as the *m*PEO-*b*-PCys-*b*-PHis hybrid terpolymers, the sequential addition synthetic approach of the corresponding anhydrides of the amino acids was used, after the completion of the polymerization of each monomer. In case of *m*PEO-*b*-[PCys-*co*-PHis] terpolymers, the macroinitiator polymerized the mixture of the two anhydrides. Then, the hybrid polypeptides were precipitated followed by deprotection of the trityl group of His by TFA. Finally, the deprotection of cysteine was achieved by using 1,4 dithiothreitol (DTT). A general reaction sequence for the synthesis of hybrid terpolymers of the general type mPEO-*b*-P(Cys)-*b*-P(His) (by the term general type we mean the three different structures with different PCys topology) is given in Scheme 2.

**Scheme 1.** Schematic representation of the synthesized hybrid terpolymers. PEO is depicted with blue color. PCys is colored magenta, while PHis is green. At the top terpolymer, the polypeptidic monomers are randomly distributed along the chain, at the middle, the PCys is located between the PEO and PHis chains, while at the lower polymer, the PCys is located at the edge of the polymeric chain.

**Scheme 2.** Reactions used for the synthesis of the fully protected polymers of the general type *m*PEO-*b*-P(Cys)-*b*-P(His). PHis has a green color, PCys is red, while PEO is blue.

Since the PEO blocks were equal for all the polymers, the code of the hybrid polypeptides was defined by the order of the blocks as well as the monomeric units of L-cysteine; therefore, the abbreviation PHis-PCys5 refers to the triblock *m*PEO227-*b*-P(His)40-*b*-P(Cys)5 and PCys10-PHis refers to *m*PEO227-*b*-P(Cys)10-*b*-P(His)35, while in case of the *m*PEO227-*b*-[P(Cys)5-*co*-P(His)40], where the polypeptidic block is composed of randomly distributed peptides, PCys5COPHis will be mentioned.

### *2.4. Self-Assembly of Empty NPs via Solvent Switch Method*

The ability of the synthesized polymers to self-assemble in aqueous media was examined at five different isotonic buffers, with different pH values and GSH concentrations. The pH values of 7.4 and 6.5 were adjusted with a PBS buffer solution (10 mM, 150 mM NaCl), while the pH 5.0 was achieved with an acetate buffer solution (10 mM, 150 mM NaCl). At pH 6.5 and 5.0, another two buffers were prepared containing 10 mM of GSH, in order to study the influence of this reducing agent in the self-assembly behavior of the NPs. In a typical procedure, 10 mg of the hybrid polypeptides, as well as 0.02 g of DTT were dissolved in 2 mL of DMSO. After the complete dissolution, 18 mL of MilliQ water were added dropwise, and the whole mixture was left under stirring overnight. The next day, the solution was placed in a dialysis membrane (Spectrapor MWCO 3500 Da) and was dialyzed against 2 L of PBS buffer pH = 7.4 for 3 h. Then, the dialysis membrane was transferred to a fresh media of the same buffer and dialyzed for another 3 h with the presence of 10 mL H2O2. The last dialysis was lasted 12 h and then the solution of the NPs was collected and divided in 5 equal parts. The first part was kept for DLS measurements, while the remaining solution was transferred equally in four different dialysis membranes (Spectrapor MWCO 3500 Da) and was dialyzed against 2 L of the following buffers for 24 h with frequent changes of the external media: PBS pH = 6.5, PBS pH = 6.5 and 10 mM GSH, pH = 5.0, pH = 5.0 and 10 mM GSH. Finally, the solution of each membrane was collected and measured with DLS, after filtration with a 0.45 μm hydrophilic filter.

### *2.5. Loading of Anticancer DOX*

In a typical experiment, 10 mg of the fully deprotected polymers was dissolved in 2 mL of DMSO and left under stirring overnight, to afford clear solutions. In case of the

polymers containing PCys, 0.02 g (0.13 mmol, 9:1 mol DTT/mol Cys) DTT was added, in order to avoid the undesirable crosslinking reactions. Subsequently, a special treatment of DOX (HCl-salt) was conducted, according to a standard procedure described by Kataoka et al. [42]. In line with this protocol, 5 mg of DOX hydrochloride was dissolved in 100 mL of MilliQ water, and the resulting red solution was added in a separatory funnel containing 100 mL of chloroform. Then, 3.0 equivalent of triethylamine (TEA) (mol Et3N: mol Dox × HCl = 3:1) was added in the aqueous phase and the color immediately turned to purple. After shaking the solution, the color became red again and the DOX was distributed in the organic phase. The concentration of DOX in the aqueous phase was estimated photometrically at 485 nm and the pH measured was close to neutral. Then, the hydrophobic DOX-free base dissolved in chloroform was separated and collected in a flask. The organic solvent was distilled off and the solid DOX was obtained. Afterwards, the solution of each polymer in DMSO was added in the flask containing the dried DOX and was left for half an hour to be dissolved. Then, 8 mL of PBS buffer (pH = 7.4) was added dropwise to the above mixture over a period of 10 min. The solution was then placed in a dialysis bag (Spectrapor, MWCO: 3500 Da, 25 ◦C) and was dialyzed against 4 L of isotonic PBS buffer at pH = 7.4 (150 mM NaCl, 10 mM PBS), in order to remove the excess drug. After 3 h of dialysis, the external buffer was renewed, and 30 mL of H2O2 was added in the fresh buffer, in the case of the polymers containing poly(L-cysteine), in order to induce the crosslinking reaction. The dialysis lasted another 3 hours and then the same procedure was repeated, without the addition of H2O2, for 12 h in total. The next day, the solution inside the membrane was obtained and the volume measured was about 12 mL. Then, about 4 mL of the NP solution was preserved for further analysis and the rest of the solution was divided into five equal parts of 1.5 mL and each part was added in a new dialysis membrane (Spectrapor, MWCO: 6000–8000 Da) and was immediately immersed in 35 mL of buffers with different characteristics, as far as the pH, the temperature and the concentration of GSH are concerned, in order to study the in vitro DOX release profile. The encapsulation efficiency (EE) and the loading capacity (LC) of the different NPs were calculated by UV absorption spectroscopy at 485 nm, as the polymer did not absorb at this wavelength, while free DOX does. Quantification was achieved by calibrating the instrument with dissolved DOX in the corresponding PBS buffer.

The encapsulation efficiency and the loading capacity were calculated according to the following equations:

EE (%) = (mass of Dox in NPs/ mass of Dox in the initial solution) × 100

LC (%) = (mass of Dox in NPs/ polymer mass) × 100

### *2.6. In Vitro Drug Release Studies*

In vitro DOX release experiments were conducted at three different pH values (pH = 7.4, 6.5 and 5.0), at two temperatures (37 ◦C and 40 ◦C) and as far as the polymers with poly(L-cysteine) in their polypeptidic block are concerned, the factor of the addition of GSH was studied. More precisely, after the completion of the dialysis procedure, the remaining solution of NPs was divided into five equal parts of 1.5 mL, as mentioned above, was transferred into a new dialysis bag (Spectrapor, MWCO: 6000–8000 Da) and was immediately immersed in 35 mL of each of the in vitro release medium. The first membrane was ingrained in a PBS buffer at pH = 7.4 and at 37 ◦C (0.010 M PBS, 0.150 M NaCl) under stirring at 200 rpm. For the release studies at the acidic pH (6.5 and 5.0), two different samples were employed, for each pH value. The first dialysis bag was immersed in a PBS buffer at pH = 6.5, at 40 ◦C, (0.010 M PBS, 0.150 M NaCl) and the other was introduced into the same release medium containing 10 mM of GSH. Similarly, in the case of the pH = 5.0, the first membrane was added in an acetate buffer at pH = 5.0, at 40 ◦C, (0.010 M acetate, 0.150 M NaCl), and the last was sank into the same buffer, at the same conditions with further addition of 10 mM GSH. The cumulative release of the drug was measured at the exterior solution at defined time intervals. The dialysis membrane was transferred into a fresh buffer solution at every

interval, in order to avoid saturation of the solution from the hydrophobic drug. The DOX concentration was calculated by UV spectroscopy at λ = 485 nm, using a calibration curve obtained with solutions of known DOX concentration measured using the same instrument.

### *2.7. In Vitro Cytotoxic Activity: Sulforhodamine B (SRB) Assay*

The established human cell lines from breast cancer MCF-7 (estrogen and progesterone receptor positive invasive ductal carcinoma), T-47D (progesterone receptor positive invasive ductal carcinoma) and MDA-MB231 (triple negative breast cancer) were used and provided by the pharmacology laboratory of NCI (National Cancer Institute, NIH, Frederick, MD, USA).

Cell culture was performed in RPMI 1640 medium (Gibco®, Code: 31870025) supplemented with 5% fetal bovine serum (FBS: fetal bovine serum, (Biosera, Code: 1001G)), 2 mM L-glutamine (Biosera, Code: XO-T1715), 100 U mL−<sup>1</sup> penicillin and 100 μg mL−<sup>1</sup> streptomycin (Biosera, Code: XO-A4122). The cell cultures were kept in an incubation oven, at 37 ◦C, in an atmosphere of 5% CO2 and 95% humidity.

The antiproliferative activity of the NPs was tested by the colorimetric method of SRB [43,44]. SRB is an anionic micromolecular compound that is stoichiometrically attached to the basic amino acid residues of protein chains, under slightly acidic conditions, and then extracted, under basic conditions.

This process involves the following steps. At the beginning of each experiment, the viability of the cells is checked with the trypan blue method so that it is always greater than 96%. The cells are added to 96-well flat-bottom cell culture plates (density 5000–10,000 cells per position) and incubated for 24 hours in an incubation oven at 37 ◦C, 5% CO2 and 95% humidity to return to the logarithmic development phase (adjustment period). After 24 h, the NP solutions are added. In some cells, only culture material is added to provide the control cells (control, C). Each NP solution was tested in four logarithmic concentrations with a maximum concentration of 10 μM. The final concentration of DMSO in each cell culture was not higher than 0.1%. A number of sites from each cell line in each experiment are fixed with 50% *v*/*v* TCA (Trichloroacetic Acid) (Applichem, Code: A1431) cold solution for 1 h at 4 ◦C, after 24 hours of the adjustment period, aiming the representation of cell culture in the phase of addition at NPs (Tz). After 48 hours of incubating the cells with the NPs, the cells are fixed by gently adding 50% *v*/*v* TCA to each site of the cell culture plate, for 1 hour, at 4 ◦C. The cells are then carefully washed, 3 times, with deionized water, the excess water is removed and the plates are allowed to dry at room temperature. The cells are stained with a solution of 0.04% *w*/*v* SRB (from SIGMA, Code: S9012) in 1% acetic acid (from Fluka, Code: 45731), for 10 minutes, at room temperature. After incubation, the excess dye is removed by repeated rinsing with 1% *v*/*v* acetic acid and the cell monolayers are allowed to dry at room temperature. A 10 mM Tris base solution is then added and the cells are incubated for 10 minutes at 37 ◦C. Under these conditions, the protein-bound dye is released into the slightly basic Tris base solution. For each concentration of the studied NP solution, the optical absorption at 540 nm (Ti) is measured on a BioTek microplate reader (Biotek, EI-311).

Using the optical absorption measurements of the cells at the time of addition of the NPs (Tz), the control cells (C), as well as the cells under the influence of the examined NPs, the percentage growth of the cells (% growth rate) can be calculated with the use of the following equations:

[(Ti − Tz)/(C − Tz)] × 100, for concentrations where Ti ≥ Tz and

[(Ti − Tz)/Tz] × 100, for concentrations where Ti < Tz

From the resulting dose–response curves (response, the cell growth rate, % growth rate) the parameters GI50, TGI and LC50 are determined, where:

GI50, Growth Inhibition 50% = the concentration of the drug through which cell growth is inhibited by 50%.

TGI, Total Growth Inhibition = the concentration of the drug through which total inhibition of cell growth is achieved.

LC50, Lethal Concentration 50% = the concentration of the drug that causes death in 50% of the cell population [45,46].

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

### *3.1. Synthesis and Characterization of the N-Carboxy Anhydrides (NCAs)*

The synthesis of the *N*-carboxy anhydrides of *α*-amino acids was monitored by FT-IR spectroscopy, while the successful synthesis and the high purity of the final monomers were confirmed by 1H-NMR spectroscopy. The results from the characterization of the *N*-carboxy anhydrides are summarized in Supplementary Materials (Figures S1–S6, Schemes S1–S3).

### *3.2. Synthesis and Characterization of the Polymers*

Initially, the novel fully protected polymers of the general type of PEO-*b*-P(*Nim*-*Trityl*-L-His)-*b*-P(*t*BM-L-Cys) were synthesized followed by the selective deprotection of each polypeptide block, to afford the fully deprotected polymers of the general type of *m*PEO*b*-P(Cys)-*b*-P(His). The synthetic procedure was monitored by FT-IR spectroscopy, and the molecular weights were obtained by using SEC-TALLS while the controlled cleavage of the protective groups was confirmed by 1H-NMR. The polymers were excessively characterized and the characterization results are shown in Table 1. It can be seen that the novel hybrid terpolymers exhibited a high degree of molecular and compositional homogeneity, while the experimentally obtained molecular characteristics were within 10% close to the stoichiometric one. In addition, the total molecular weight of the polypeptidic blocks were close in all polymers although the ratios between the PHis and PCys were different, while PEO was the same.


**Table 1.** Molecular characteristics of the hybrid terpolymers of the general type of *m*PEO-*b*-P(Cys)-*b*-P(His).

<sup>a</sup> *M*<sup>n</sup> PEO. <sup>b</sup> Experimental *M*<sup>n</sup> of deprotected P(Cys)x-P(His)y blocks obtained by SEC-TALLS subtracting the MW of the PEO block. Measurements were conducted using 0.10% TFA (*v*/*v*) solution of H2O/ACN (80/20 *v*/*v*) as the eluent at 35 ◦C.

As an example, we will present the characterization of the PCys5-PHis. The FT-IR spectra of the block copolymer *m*PEO227-*b*-P(*t*BM-L-Cys)5-*b*-P(*Nim*-*Trityl*-L-His)40 is presented in Figure S22. Spectrum A corresponds to the protected copolymer *m*PEO227 *b*-P(*t*BM-L-Cys)5. The vibration at 1637 cm–1 is attributed to the C=O bond of the amide bond. Other characteristic peaks appear at 1100 cm–1 and 2890 cm–1, which are due to the amplitude vibration of the ether bond C–O–C of PEO and C–H bonds respectively. In addition, the vibration at 1740 cm–1 corresponds to the vibration of one carbonyl group of L-cysteine *N*-carboxy anhydride, which indicates that the polymerization of the first monomer has not been completed at the time of the measurement. Spectrum B (Figure S22) corresponds to the copolymer *m*PEO227-*b*-P(*t*BM-L-Cys)5-*b*-P(*Nim*-*Trityl*-L-His)40 and was obtained approximately 14 days after the addition of the second monomer (*Nim*-*Trityl*-L-His NCA). In this spectrum, the characteristic peak at 1679 cm–1 is observed, which corresponds

to the amide bond, as well as the absorption bands at 1106 cm–1 of PEO. Additional peaks that appear in this spectrum are the vibration at 1784 cm–1, which is due to the one carbonyl group of *Nim*-*Trityl*-L-His NCA, and indicates that the polymerization of the PHis block has not yet been completed. Additionally, the peaks at 745 cm–1 and 703 cm–1 are attributed to the bending vibrations of the -CH=CH- bonds of the aromatic rings of the *trityl* protecting groups of PHis. Spectrum C corresponds to the final fully protected block copolymer, which was isolated after precipitation in diethyl ether. This spectrum shows exactly the same absorption bands as spectrum B, with the difference that the vibration at 1784 cm–1 of the anhydride is absent, as the histidine monomer is completely consumed. Finally, the spectrum D corresponds to the desired fully deprotected PCys5PHis. In this spectrum the vibrations at 746 cm–1 and 702 cm–1, which correspond to the *trityl* protecting groups of the poly(L-histidine), are absent, proving the successful deprotection of this polypeptide block. However, there are no accurate data from the FT-IR spectrum to verify the successful deprotection of poly(L-cysteine) building blocks, as the vibration signals of -SS-, -CH2-S-, -SH, -CH (*t*-butyl) bonds are very weak. The successful synthesis and the purity of the terpolymer was confirmed by 1H-NMR spectroscopy in D2O/DCl 1% solvent, after each deprotection step. It is observed that all peaks in both spectra (Figure S23) are attributed to the hydrogens of the polymer. In Figure S23, the upper spectrum corresponds to the histidine-deprotected *m*PEO227-*b*-P(*t*BM-L-Cys)5-*b*-P(His)40, while the second is attributed to the final fully deprotected PCys5PHis. Figure S23, Spectrum A: 1H-NMR (600 MHz, D2O/DCl 1%, δ, ppm): 1.34 (i: 9H, (CH3)3–C–), 3.18–3.24 (f + g: 4H, –CH2–), 3.41 (h: 3H, CH3–O–), 3.35–3.90 (e: 4H, –CH2–CH2–O–), 4.40 (d: 1H, NH–CH(CH2–S–S)–C=O), 4.77 (c: 1H, NH–CH(CH2–Im)–C=O), 7.35 (b: 1H, –C=CH–N–), 8.71 (a: 1H, –N=CH–N–). Figure S23, Spectrum B: 1H-NMR (600 MHz, D2O/DCl 1%, δ, ppm): 1.35 (i: 9H, (CH3)3–C–), 3.20–3.24 (f + g: 4H, –CH2–), 3.42 (h: 3H, CH3–O–), 3.35–3.90 (e: 4H, –CH2–CH2–O–), 4.53 (d: 1H, NH–CH(CH2–S–S)–C=O), 4.75 (c: 1H, NH–CH(CH2–Im)–C=O), 7.36 (b: 1H, –C=CH– N–), 8.71 (a: 1H, –N=CH–N). Finally, the histidine-deprotected terpolymer *m*PEO227-*b*-P(*t*BM-L-Cys)5-*b*-P(His)40 was characterized by SEC chromatography in H2O/TFA solvent (Figure S24).

A similar procedure was followed for the synthesis and characterization of all hybrid terpolymers. The characterization results from all polymers obtained by FT-IR, 1H-NMR and SEC are presented in Supplementary Materials (Figures S7–S30).

### *3.3. Secondary Structure through Cyclic Dichroism*

It is well known that polypeptides have the ability to mimic natural proteins by adopting secondary structures in response to various external stimuli (temperature, pH, etc.).

In order to investigate their structural and conformational changes by pH and temperature, we studied the synthesized polymers by CD. More precisely, CD measurements were conducted at four different pH values: pH = 7.4 (pH of the healthy tissue), pH = 6.5 (pH of the extracellular environment of the tumor tissue as well as early endosome pH within the cells), pH = 5.0 (pH of the lysosomes within the cell) and pH =3.0, and at three different temperatures: 25 ◦C (room temperature), 37 ◦C (temperature of the healthy tissue) and 40 ◦C (temperature of cancer tissue). We studied the PCys-protected polymers, while only PHis was deprotected, in order to avoid crosslinking.

In most cases, the results revealed a similar conformational transition of the secondary structure from a beta turn at higher pH values (pH = 7.4 and pH = 6.5) to a random coil conformation at lower pH values (pH = 5.0 and pH = 3.0), as shown in Figure 1 as well as Figures S31–S34. The negative peaks at 190 in combination with the positive peak at 205 nm and a slight negative peak at 218 nm are indicative of the *β*-turn type 2 conformation [47], the negative peak at 218 and a positive at 195 nm reveal a *β*-sheet conformation, while the negative peak at 225 nm is indicative of an *α*-helix conformation. At lower pH, the negative peak at 196 nm in combination with the positive peak at 218 nm are characteristic of the random coil structure.

**Figure 1.** CD spectra at different pH values, at 25 ◦C: (**a**) *m*PEO227-*b*-P(*t*BM-L-Cys)5-*b*-P(His)40, (**b**) *m*PEO-*b*-(P(His)35-*co*-P(*tBM*-L-Cys)10), (**c**) *m*PEO227-*b*-P(*t*BM-L-Cys)10-*b*-P(His)35, (**d**) *m*PEO-*b*-P(His)44.

As we showed in our previous work, at higher pH, the *β*-turn is a conformation that enthalpically favors the structure of PHis homopolymer [48]. In this conformation, the imidazole rings come close, developing the maximum hydrogen bonds. In this structure, a loop is created every three amino acids, since the nitrogen of the imidazole ring of an amino acid forms hydrogen bonds with the carbonyl group of the following amino acid and at the same time, forms hydrogen bonds with the hydrogen of the imidazole ring of the following amino acid. At lower pH, the secondary structure changes from *β*-turn to random coil conformation.

The conformational transitions obtained by altering the pH (*m*PEO227-*b*-P(*t*BM-L-Cys)10-*b*-P(His)35 (Figure 1c), *m*PEO227-*b*-P(His)40-*b*-P(*t*BM-L-Cys)5, *m*PEO227-*b*-P(His)35 *b*-P(*t*BM-L-Cys)10 and *m*PEO227-*b*-[P(*t*BM-L-Cys)5-*co*-P(His)40]) (Figure S32–S34, see supporting information) are similar to the one obtained by PEO-*b*-PHis diblock copolymer (Figure 1d). At the triblocks, a lower pH is required as compared to the one required for PEO-*b*-PHis to achieve the transition to the random coil conformation, due to the higher amount of hydrophobic blocks. At these terpolymers, the absorption of the PHis block dominates and overlaps the absorbance of the *β*-sheet conformation of the protected PCys block. However, in some cases, the secondary structure of the *β*-sheet is evident, which is the typical conformation of the free and protected PCys [49]. The terpolymer PEO-*b*-P(*t*BM-L-Cys)5-*b*-P(His)40 (Figure 1a) at pH = 7.4 exhibits a mixed structure of *β*-turn and *β*-sheet, which is attributed to both the PHis and PCys moieties. At pH = 6.5, a mixed structure of *β*-turn and *α*-helix is observed, as we can see a negative peak at 190 and a positive at 205 nm, while we also observe a negative peak at 230 nm indicative of the *α*-helix conformation. Finally, at more acidic pH (pH = 5.0 and pH = 3.0), only the conformation of the random coil is observed.

In case of *m*PEO-*b*-(P(His)35-*co*-P(*tBM*-L-Cys)10) (Figure 1b), the presence of a larger amount of PCys randomly distributed along the PHis chain induces a larger amount of *β*-sheet conformation at neutral pH. This is more pronounced at this copolypeptide due to the higher amount of PCys (Figure 1b) rather the one with lower amount and the same structure (random distribution of PCys) (Figure S34). It seems that the small amount of PCys did not have significant impact on the secondary structure. The difference of the secondary structure obtained from the random as compared to the block copolypeptides is proof of the random distribution of PCys along the PHis chain on the PCys5COPHis and PCys10COPHis terpolymers. Finally, it was found that by increasing the temperature maintaining a constant pH, from 25 ◦C, to 37 ◦C and then to 40 ◦C, the conformation is not altered (Figure S31), which shows that the polymers do not show a temperature responsiveness.

### *3.4. Self-Assembly of the Empty Hybrid Polymers*

The ability of the synthesized polymers of the general type *m*PEO-*b*-P(Cys)-*b*-P(His) to form nanostructures was achieved via a solvent switch method, by applying the dialysis technique, with the use of DMSO as the common good solvent and aqueous solution at pH = 7.4, as the final media. During this procedure, a simultaneous crosslinking reaction was conducted, using H2O2 as the oxidative agent to form the disulfide bonds which stabilize the NPs. The ability of the polymers to self-assemble as well as the structural characteristics of the NPs were examined by DLS, SLS and TEM. At pH = 7.4, in which the self-assembly and crosslinking takes place, two populations are always observed by DLS (Figures S35–S39). The average size of the small population is about 30 nm, while the larger population is about 250 nm. The appearance of two populations is also observed by TEM microscopy. More specifically, for the crosslinked polymer PCys5-PHis the TEM image depicted at Figure 2g shows small spherical and elliptical vesicles within the core of a larger spherical nanostructure. The large NPs are composed of a large core containing multiple small vesicles. The matrix of the core is a mixture of PHis and PCys. Therefore, the dimensions of the NPs obtained by DLS (~31 nm, Figure S38) are the small vesicles within the core of the large NPs shown as the second population. The core of the NPs obtained by TEM for these NPs is almost 210 nm but if we add the PEO corona, we will achieve dimensions close to 220 nm which are smaller than the 250 nm obtained by DLS (Figure S35). This is probably due to the different processes followed for the sample preparation for DLS and TEM. For DLS, the nanoparticles are dissolved in PBS buffer pH = 7.4, while for TEM imaging, the treatment includes the removal of the salts by dialysis, freeze drying and finally redissolution in MilliQ water to be placed on the grid and the final evaporation of water to dryness. This difference in dimensions obtained between the two methods is common in many works [28], where TEM gives smaller dimensions, and can be attributed to the shrinkage caused by the evaporation of water. No TEM measurement was performed for the crosslinked polymer PCys10-PHis, as it precipitated during the process of self-assembly and crosslinking at PBS buffer pH = 7.4. The precipitation is due to the increased hydrophobicity and crosslinking, which is a consequence of the higher percentage of PCys in the polypeptide block and the close packing of PCys since they are obliged to be organized and located at the interphase between the PEO and PHis phases. In all cases, TEM measurements (Figure 2) revealed spherical micellar structures, with a multivesicular core comprised of PHis and PCys polypeptides and a hydrophilic corona of PEO. This kind of self-organization is consistent with the results from DLS and SLS, as mentioned.

SLS measurements confirm the self-assembly of the NPs in structures containing a multivesicular core, as the ratio Rg/Rh is close or slightly larger than 1. Table 2 summarizes the polymers of the present work and the corresponding values of the sizes Rg, Rh and Rg/Rh, at pH = 7.4, at 25 ◦C.

Finally, a general observation concerning all NPs is that in TEM images, around the gray core, a faint, white crown can be seen, which is attributed to the PEO block, as it does not create a strong contrast. This phenomenon comes in agreement with the results from z-potential measurements (Table 2), which reveal that at pH = 7.4, the mean value of the z-potential is in the range [−6.8 mV, + 3.3 mV], therefore, all the synthesized NPs have

in most cases a neutral surface charge, indicating that the PEO block consists of the outer periphery of the nanostructures.

**Figure 2.** TEM image of the empty crosslinked NPs of (**a**) PCys5COPHis (scale bar is 0.5 μm); (**b**) histogram of size distribution of NPs of (**a**); (**c**) PHis-PCys10 (scale bar is 0.10 μm); (**d**) histogram of size distribution of NPs of (**c**); (**e**) PCys10COPHis (scale bar is 0.2 μm); (**f**) histogram of size distribution of NPs of (e); (**g**) PCys5-Phis (scale bar is 0.2 μm); (**h**) PHis-PCys5 (scale bar is 0.1 μm); (**i**) illustration of a NP featuring a multivesicular core.

**Table 2.** Molecular characteristics of the empty NPs by DLS and TEM.


<sup>a</sup> The population with the largest dimension used was obtained by DLS.

### *3.5. pH and Redox Responsiveness of the Empty NPs*

In order to investigate the pH and redox responsiveness of the synthesized NPs at both healthy and cancerous tissue conditions, different aqueous solutions of the polymers were prepared and measurements took place at different pH values and GSH concentrations. More specifically, DLS measurements were conducted to the solutions of the empty crosslinked polymers, resulting from the dialysis process, at pH = 7.4 (pH of human blood and healthy tissues), at pH = 6.5 (extracellular pH of cancer cells and early endosomes inside the cells) and at pH = 5.0 (lysosomal pH inside the cells), as well as at pH = 6.5 and pH = 5.0 with the addition of 10 mM GSH (intracellular GSH of cancer cells).

DLS measurements revealed responsiveness towards pH and GSH concentration. In the case of PCys5-PHis NPs (Figure S35), at pH = 7.4, the results indicate the existence of two populations, one small of about 31 nm and a larger one of about 250 nm. With a decrease in pH from 7.4 to 6.5 and 5.0 (25 ◦C, 90◦), a slight increase in the diameter is observed, due to swelling of PHis through interaction with water, since at this pH PHis is protonated, which in turn leads to an increase in its hydrophilicity.

In the presence of 10 mM GSH at the acidic pH, the NPs exhibit a further redox response. GSH acts as a reducing agent and causes the cleavage of the disulfide bonds. The concentration of GSH is about 10–20 mM in cancer cells, while it is about 2 μM in healthy tissues. It is observed that at pH = 6.5, in the presence of GSH, a third population appears at 7 nm, most likely due to the rupture of the NPs to smaller particles or even single chains. In addition, at pH = 5.0 in the presence of GSH, except for the third population that appears at 7 nm, there is an additional increase in the size of the larger population from 285 nm (pH = 5.0, without GSH) to 427 nm (pH = 5.0, with GSH). Both of these results prove the synergistic response of PHis and PCys, through the variation in pH and GSH under healthy and cancerous conditions. The same trend is observed for all the polymers and the results from DLS measurements are summarized in Supplementary Materials (Figures S35–S39).
