*3.6. Self-Assembly of the DOX-Loaded NPs*

The encapsulation of DOX was performed on all series of the polymers, as well as on another two polymers PEO227-*b*-P(His)44 and PEO227-*b*-P(Sar)98-*b*-P(Cys)30. DLS and TEM techniques were employed to obtain the structure and the morphological characteristics of NPs, while z-potential measurements were conducted to determine their surface charge. All the results are summarized in Table 3 and Figure 3, while DLS results are presented in Supplementary Materials (Figures S40–S43).


**Table 3.** Molecular characteristics by DLS and TEM, encapsulation efficiency and loading capacity of the DOX-loaded NPs.

Surprisingly, the results from DLS and TEM revealed that in some cases, the DOXloaded NPs can self-assemble into homogeneous core–shell micellar structures, instead of polydisperse micellar structures with a multivesicular core obtained by the empty one. It can be seen (Table 3) that the diameter of the NPs obtained by TEM is smaller than the Rh, due to the different preparation methods followed, as referred to previously. As an example, the TEM images and the size distribution of the core of the NPs obtained by the PCysX-PHis, are shown in Figure 3b–d. It is obvious that the NPs formed by these terpolymers are

core–shell micelles, composed of a core containing the hydrophobic polypeptides along with encapsulated DOX, with a corona composed of a PEO chain and water. The dimension of the NPs obtained by DLS is close to 190 nm, while the one obtained by TEM is close to 110 nm. In case of the TEM images, we see only the core of the NPs, while by DLS we see the outer dimensions of the NPs. Still, the addition of the PEO which is smaller than 10 nm on the dimensions obtained by TEM cannot match the dimensions of the DLS. The structure of the micelles is illustrated in Figure 3n.

**Figure 3.** TEM image of the DOX-loaded NPs of (**a**) PCys5COPHis (scale bar is 0.5 μm); (**b**) PCys10- PHis (scale bar is 0.1 μm); (**c**) PCys5-Phis (scale bar is 0.5 μm); (**d**) histogram of size distribution of NPs of (**c**); (**e**) PCys10COPHis (scale bar is 0.2 μm); (**f**) PHis-PCys10 (scale bar is 0.1 μm); (**g**) histogram of size distribution of NPs of (**f**); (**h**) PHis-PCys5 (scale bar is 0.2 μm); (**i**) histogram of size distribution of NPs of (**h**); (**j**) *m*PEO22-*b*-P(His)44 (scale bar is 0.2 μm); (**k**) histogram of size distribution of NPs of (**k**); (**l**) *m*PEO227-*b*-P(Sar)98-*b*-P(Cys)30 (scale bar is 0.2 μm); (**m**) cartoon of a micellar structure composed of a multivesicular core loaded with DOX; (**n**) cartoon of a core–shell micelle, with the core loaded with DOX; (**o**) cartoon of a vesicular structure with the polymeric monolayer loaded with DOX. PHis has a green color, PCys is magenta, while PEO is blue. The vesicles at the core at Figure 3 (**m**) is a mixture of PHis (green) with PCys (magenta).

The TEM images of the DOX-loaded NPs of the PHis-PCysX as well as the size distribution of the core of the NPs are shown in Figure 3f–i. The NPs are vesicular structures of rather small dimensions. In these images, a faint white diffuse cloud is observed that surrounds the vesicle and is due to the hydrophilic block of PEO. The possible structure of the vesicles is illustrated in Figure 3o. Particularly at these two polymers, the significant difference between the DLS and TEM dimensions is probably due to the formation of vesicular structures, where the elimination of the solvent is expected to result in a significant reduction in dimensions (as observed by TEM) due to the shrinkage of the NPs.

The TEM images of the DOX-loaded NPs of PCysXCOPHis are depicted on Figure 3a,e. The TEM image of PCys5COPHis in Figure 3ashows ruptured aggregates, probably ruptured vesicular structures. It seems that the small amounts of PCys at this polymer did not result in efficient crosslinking that would stabilize the aggregate. It seems that the random distribution of the small amount of PCys did not result in efficient crosslinking. On the contrary, in the case of the PCys10COPHis with higher amount of Pcys (Figure 3e), the NPs were more robust and aggregates with a core containing smaller vesicles were formed. The vesicular structures within the core were very small and could not be distinguished by DLS. In that series of NPs, the dimensions obtained by DLS and TEM are close.

The TEM image and the core size distribution of the DOX-loaded NPs of *m*PEO227-*b*-P(His)44 are shown in Figure 3j,k, respectively, while the TEM of the *m*PEO227-*b*-P(Sar)98-*b*-P(Cys)30 is shown in Figure 3l. Both NPs self-assemble into spherical core–shell micellar structures. The hydrophobic core of the micelle, where DOX is encapsulated, consists of the blocks of PHis and PCys for the polymers *m*PEO227-*b*-P(His)44 and *m*PEO227-*b*-P(Sar)98 *b*-P(Cys)30, respectively. The outer hydrophilic corona of the micelles is attributed to the PEO block in the case of *m*PEO227-*b*-P(His)44 and to the PEO and poly(sarcosine) blocks for the polymer *m*PEO227-*b*-P(Sar)98-*b*-P(Cys)30. It is obvious that the presence of PCys at the terpolymers significantly altered the structure of the NPs as compared to the one that lacked the PCys layer.

Finally, Z-potential measurements were conducted in all DOX-loaded NPs, in order to determine their surface charge. The results are presented in Table 3 and show that at pH = 7.4, the mean value of the z-potential is in the range [−8.1 mV, + 1.1 mV], concluding that all synthesized NPs have a neutral surface charge. These results come in accordance with the observations from TEM images, which prove that the uncharged and hydrophilic block of PEO is located at the outer periphery of the nanoparticle. In summary, TEM imaging revealed how the PCys topology as well as the encapsulation of DOX affects the morphology of the DOX-loaded NPs. Thus, it is expected that the topology of the polypeptidic blocks will influence the kinetics of drug release under healthy and cancer cell conditions.

### *3.7. Drug Loading and In Vitro Release Studies*

Drug loading was performed at pH = 7.4, using PBS isotonic buffer (150 mM NaCl, 10 mM PBS). The encapsulation efficiency (EE) of the drug and the loading capacity (LC) of the various NPs were determined by UV–Vis spectrophotometry at 485 nm, since only DOX absorbs in this wavelength. Quantification was performed using a standard DOX calibration curve in the corresponding PBS buffer pH = 7.4, presented in Supplementary Materials (Figure S44). Table 3 summarizes the results from UV–Vis spectroscopy measurements.

The drug release profile was examined at various pHs, temperatures, and in the presence of GSH, in order to simulate the release conditions in both healthy (pH = 7.4, 37 ◦C) and cancer tissue (pH = 6.5, 40 ◦C, 10 mM GSH) as well as late lysosomes environment of the cancer cells (pH = 5.0, 40 ◦C, 10 mM GSH). The amount of DOX released was determined by UV–Vis spectrophotometry at 485 nm and quantification was performed using standard calibration curves of the drug in the respective buffers, presented in Supplementary Materials (Figures S45 and S46).

It can be seen that the NPs consisting of the aggregated polymer PCys5-PHis in Figure 4a are pH-stimuli responsive, since after 144 hours, 34% of the drug has been released at pH 7.4, 56% at pH 6.5 and 60% at pH 5.0. It is obvious that as the pH of the release medium decreases, the percentage of released DOX increases. This effect is expected, since at acidic pH, the imidazole ring of histidine is protonated, rendering the PHis blocks hydrophobic, leading to the swelling or the rupture of NPs. This is in agreement with the DLS results. Finally, the rupture of the nanoparticles leads to the release of DOX in a pH-controlled manner. It seems that PCys is not contributing significantly to the release of the drug, since the lowering of only the pH results in a significant increase at the release rate of DOX. Therefore, 5 monomeric units of Cys is not enough to create a strong crosslinked layer that will direct the release of the drug.

**Figure 4.** Cumulative release of the DOX-loaded NPs: (**a**) PCys5-PHis, (**b**) PCys10-PHis, (**c**) PHis-PCys10, (**d**) PEO-*b*-PHis, (**e**) PEO-*b*-(PSAR)98-*b*-(PCys)30, (**f**) RPMI.

Contrary to the NPs formed by PCys5-PHis with the lower amount of PCys, the NPs formed by the polymers exhibited the same architecture but higher PCys amount, i.e., PCys10-PHis, at 144 hours, only 15% of the drug was released at pH = 7.4, 29% at pH = 6.5, while at pH = 5.0, 79% was released (Figure 4b).

It is worth noting that the percentages of the drug released at pH = 7.4 and 6.5 from the PCys10-PHis NPs are lower obtained at all the NPs. This may be due to the presence of the crosslinked PCys layer at the interphase of PEO that maintain the cargo within the core until it is heavily ruptured by an increased concentration of GSH (see Figure 3n). At the same time, the greater stability of the NPs due to the more extensive crosslinking leads to a more pronounced response to the GSH concentration, as at pH = 6.5 without GSH, the release reaches 29%, and at the same pH in the presence of GSH, the percentage increases to 45%. At pH = 5.0, without GSH the release reaches 75%, while at pH = 5.0 in the presence of GSH, it reaches 84%, demonstrating a strong synergistic response to these stimuli (pH and GSH). It seems that the strong swelling of the core at pH = 5.0 even without GSH leads almost to the rupture of the PCys crosslinked layer.

For the DOX-loaded NPs of PHis-PCys5 hybrid terpolymer, pH plays a more critical role than GSH. The NPs formed are micellar structures with a multivesicular core that are expected to be less robust than the core–shell micelles. As in the case of the PCys5-PHis, by lowering the pH we have a significant release of the drug (Figure S47). Due to the formation of a PCys monolayer within the bilayer of the hybrid copolypeptide (see Figure 3o), the release of the drug at pH = 7.4 remains rather low (about 25%) but increases significantly by the protonation of PHis when the pH is lowered. This can be attributed to the transition of PHis from hydrophobic to hydrophilic, and since most of the drug exists within the PHis layer, this switch leads to increased release.

In case of PHis-PCys10 NPs, it was found that the vesicles formed release a significant amount of drug at pH = 7.4. The release become gradual by lowering the pH as well as the increased concentration of GSH. In this case both parameters contribute equally. The maximum cumulative release was 65% (Figure 4c).

For the DOX-loaded PCys5COPHis NPs, core–shell structures composed of a multivesicular core were probably formed that were not robust as indicated by the rupturing under vacuum to dryness (Figure 3a). We have a significant release of drug even at pH = 7.4 which increases gradually by the lowering of pH (Figure S48) and the addition of GSH. The maximum cumulative release obtained was 75%. This release profile is similar to the one of PHis-PCys10, where both stimuli, pH and redox, contribute equally.

Slower release was obtained by the NPs formed by PCys10COPHis hybrid copolypeptides (random structure) (Figure S49), due to the presence of a larger amount of PCys that hinders the release of the drug. The NPs have a similar core–shell structures exhibiting a multivesicular core similar to the one with the lower amount of PCys and the same structure. Although the release at pH = 7.4 is rather high, there is a gradual increase in the release by lowering the pH and addition of GSH. Similar gradual release profile was obtained by the vesicular structures of PHis-PCys10 as well as PCys5COPHis NPs.

In order to elucidate the influence of the PCys on the release profile of the loaded NPs, we studied the encapsulation and release of DOX of the NPs obtained by the hybrid copolypeptides *m*PEO227-*b*-P(His)44 and *m*PEO227-*b*-P(Sar)98-*b*-P(Cys)30 shown in Figure 3d,e, respectively. The DOX loaded NPs of the polymer *m*PEO227-*b*-P(His)44 are core–shell micelles. They show responsiveness only to pH due to the PHis block. Thus, at 144 hours 35% of the drug has been released at pH = 7.4, 63% at pH = 6.5 and 75% at pH = 5.0. It is obvious that although the cumulative release at pH = 7.4 is comparable with most of the NPs in this work, at pH = 6.5 and 5.0 the release is higher. The lower release rates of the DOX at the terpolymers is due to the contribution of the hydrophobic PCys and its crosslinking. At pH = 7.4 where both polypeptides are hydrophobic, the release profile do not depend on the presence of PCys significantly, unless PCys is located at the interphase of PEO. At lower pH, the NPs from *m*PEO227-*b*-P(His)44 lose their structure faster than the one containing PCys due to the crosslinks formed by this amino acid that stabilize the structure and a lower pH is required to reach the same release rate.

In order to compare the release profile of the terpolymers with the one containing only a hydrophilic polymer and PCys, we synthesized many block polypeptides, initially PEO-*b*-PCys44. However, the polymer was not soluble, and we found that the best solubility was on the triblock terpolymer *m*PEO227-*b*-P(Sar)98-*b*-P(Cys)30. The increase in PEO did not result in significant enhancement of the solubility of the NPs, and we incorporated PSar for that purpose. It was found that the NPs formed by the *m*PEO227-*b*-P(Sar)98-*b*-P(Cys)30 hybrid copolypeptide showed a significant release even at pH = 7.4 and inability to maintain the cargo even at neutral pH. In addition, the response to GSH was very strong, while to pH, it was minimal. The weak dependence of the release to pH is due to the protonation of DOX at lower pH which renders it more hydrophilic and not to the polymer.

In order to further examine the influence of the complex media of RPMI + FBS that mimic an even closer environment at the blood compartment, we performed drug release profiles in this media. To our knowledge, this is the first time that release curves have been performed at the cell culture medium and not only in buffers (Figure 4f). It is obvious that the release profiles are similar to that of the isotonic PBS buffer at pH = 7.4. The results show that even after 2 days, most of the drug is still encapsulated into the NPs and thus, the delivery of the drug is directed by the carriers.

### *3.8. In Vitro Cytotoxic Activity*

The antiproliferative activity of the various NP solutions was tested by the colorimetric method of sulforhodamine B (SRB, Sulfurhodamine B).

From the three cell lines tested, the most sensitive to both DOX and nanoformulations were found to be MCF-7 cells, followed by T-47Ds, while MDA-MB231 were found to be the least sensitive under the experimental conditions used (Figure 5 and Table 4). Interestingly, DOXIL (or CAELYX) does not work well in these experimental conditions, which is probably explained by its composition. In contrast to DOXIL, the four nanocarriers tested showed similar activity to DOX, as shown by both the three cell line growth curves (Figure 5) and the GI50, TGI and LC50 parameters (Table 4). The *m*PEO227-*b*-P(His)44 NPs showed a slightly better effect on MB231 cells at a concentration of 1 μM (Figure 5); however, the other four nanocarriers did not exhibit any specificity in the cytotoxic activity, similarly to free DOX (Table 4). Finally, none of the empty nanocarriers tested in the same cell lines and experimental conditions showed toxicity.


**Table 4.** GI50 (Growth Inhibiting concentration 50), TGI (Total Growth Inhibition) and LC50 (Lethal Concentration 50) against three established human breast cancer cell lines. All values are in μM.

*3.9. Influence of the PCys Topology on Self-Assembly, DOX Loading, In Vitro Release Profile as Well as In Vitro Cytotoxic Activity*

From the systematic study of the series of hybrid polymers, it is obvious that the topology of PCys plays a critical role in the structure of the NPs formed and thus, the release profile of the drug.

Concerning the empty NPs, the self-assembly resulted in the formation of micellar NPs composed of a multivesicular core and a shell from PEO chains.

**Figure 5.** Growth rate curves for (**a**) the breast cancer cell lines MCF-7, (**b**) the breast cancer cell lines MB231, (**c**) the breast cancer cell lines T47D. Negative values of growth rate denote cytotoxic activity (see Section 2.7 under Materials and Methods for the calculation of the growth rate).

The loaded NPs with DOX presented differently structured NPs as compared to the empty one, which differs depending on the topology of PCys. In the case that PCys is at the interphase between the PEO and PHis, the formed loaded NPs are core–shell micelles. The crosslinked PCys interphase tightly close the drug within the core preventing its leakage at healthy tissue conditions. The drug is released slowly at extracellular cancer pH conditions, while it is released fast and efficiently under intracellular cancer cell conditions, where we have a combination of low pH and high concentration of GSH. Under intracellular healthy conditions, the release is slower than intracellular cancer cell conditions, but slightly higher than under extracellular healthy conditions.

When PCys is at the edge of the polymeric chain, PCys which is more hydrophobic than PHis at neutral pH, interacts with DOX directing the aggregation. So, the chains aggregate first through the PCys end block creating a bilayer with two PEG hydrophilic layers at the outer part leading to the formation of vesicular structures. As illustrated in Figure 3o, the PCys (magenta) layer aggregates in an antiparallel manner to form the bilayer, leading to the formation of vesicular structures. However, due to the encapsulation of DOX also at the PHis layer, we have a significant initial release even at higher pH values, since the crosslinked PCys layer does not hinder the leakage of the drug as in the case when

PCys was at the interphase between PEO and PHis (Figure 3n). Still, the release is gradual and responds at both stimuli (pH and redox) when the amount of PCys is higher.

When the PCys is randomly distributed along the PHis chain, the formed loaded NPs are core–shell structures with a multivesicular core. They present a significant initial release at higher pH as in the case of the NPs where the PCys was at the edge of the polymeric chain and the amount of PCys was large. In that case, PCys do not form a crosslinked tight layer, since the monomeric units of Cys are not close together.

This work shows that it is possible to select a drug release profile and the structure of the NPs formed by altering the topology of PCys. In most cases, both stimuli were participating at the release profile of the NPs. Slow release can be achieved by placing PCys at the interphase of the NPs and will be released fast when the NPs reach an intracellular cancer cell environment. When PCys is located at the edge of the polymeric chain, the NPs will form vesicular structures and it will be possible to encapsulate both a hydrophobic drug within the bilayer and a hydrophilic drug at the empty interior. The release will be performed in a gradual way, by lowering the pH and increasing GSH concentration. In that case a significant amount of PCys has to be incorporated at the NPs.

A similar gradual release can be achieved when PCys is randomly distributed along the PHis chain. This release profile can be achieved even for low amounts of PCys and through a core–shell micelle structure exhibiting a multivesicular core.

Usually, the nanoparticulate drug delivery results in a higher cancer cell growth rate as compared to the corresponding growth rate of the free drug [50]. In our work, the cell culture results showed that the GI50 of the NPs is comparable or better to the free drug after two days, although at least half of the drug is still encapsulated within the NPs. In the case of the PEO-*b*-Phis hybrid copolymer, the efficacy against all cancer cell lines was even better than free DOX. This shows that the presence of PHis favors the efficient accumulation of the drugs within the cancer cells through the rupturing of the endosomes by the "proton sponge mechanism", improving the delivery of the drug within the cells. These results are very encouraging for these materials to be used as drug delivery carriers for anticancer agents.

### **4. Conclusions**

In this work, three series of novel hybrid amphiphilic terpolymers have been synthesized from the general type *m*PEO-*b*-P(Cys)-*b*-P(His) exhibiting different PCys topology, i.e., either between the PEO and PHis blocks, at the end of the polymeric chain or randomly distributed along the PHis chain. The terpolymers self-assemble to afford empty NPs mainly exhibiting the core–shell micellar structured NPs with multivesicular core. The polymeric materials can encapsulate the anticancer drug DOX to result in NPs exhibiting pH and redox responsiveness due to the PHis and PCys moieties, respectively, while PEO is always at the outer periphery presenting "stealth" properties, as z-potential measurements revealed. The encapsulated DOX was released in a controlled manner upon both stimuli, pH and GSH concentration. Depending on the PCys topology, NPs with different structures as well as release profiles were achieved. When the PCys is in the middle of the polymeric chain, core–shell micelles are formed, while the crosslinked PCys layer do not allow the leakage of the drug under healthy pH and GSH conditions. When the PCys is at the edge of the chain, vesicular structures are formed with gradual release of DOX depending on both stimuli. Finally, when PCys is randomly distributed, less robust core–shell micellar structures with a multivesicular structured core are formed that present a gradual release of the drug concerning both stimuli. The antiproliferative activity of these "smart" DOXloaded NPs was tested in three breast cancer cell lines (MCF-7, T-47D and MDA-MB231) and the results revealed similar activity to DOX. Doxorubicin continues to be a cornerstone of anticancer chemotherapy being first line drug in different types of cancers and is probably the most commonly prescribed anticancer drug. However, doxorubicin suffers from severe side effects, the development of drug-induced toxicity, mainly cardiotoxicity, and the development of drug resistance being the most significant [51,52]. The cardiotoxicity

is dose-dependent and the dose-limiting side effect of the drug may even result in the withdrawal of doxorubicin from the chemotherapeutic regimen. Drug resistance against doxorubicin and anthracyclines in general often occurs via the upregulation of MDR (Multi Drug Resistant) genes that control three different types of efflux proteins, pumping out of the cells, thus the drug reducing its intracellular concentration and ultimately diminishing its anticancer efficacy. To address these important clinical drawbacks of doxorubicin and because of the importance of this drug in oncology, drug delivery systems have been employed. The efforts so far have led to the development and clinical use of only two such systems: the liposomal doxorubicin and the pegylated liposomal formulation, while several other efforts have failed to advance such systems in the clinics. Thus, in this context, and if the subsequent evaluation of these "smart" systems show promise in addressing these pitfalls of the drug in vitro and most importantly in vivo in animal models of cancer, these materials could be very promising candidates in cancer treatment.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/pharmaceutics15030790/s1. Materials used and details on the synthesis of N-carboxy anhydrides are given in Schemes S1–S3, Figures S1–S6; details on the synthesis and characterization of the polymers are given in Scheme S4–S5, Figures S7–S30; Circular Dichroism results are shown in Figures S31–S34, DLS results are shown in Figures S35–S43; in vitro DOX release studies are shown in Figures S44–S46; release curves of the loaded nanoparticles are given in Figures S47–S49. References [53–57] are cited in the supplementary materials.

**Author Contributions:** Conceptualization, H.I.; bibliographic search, H.I., D.S. (Dimitra Stavroulaki), V.A., I.K., D.S. (Dimitrios Skourtis), P.G.F., A.L., D.K. and K.D.; methodology, D.S. (Dimitra Stavroulaki), V.A., I.K., D.S. (Dimitrios Skourtis), P.G.F., A.L., S.S., G.P. and P.T.; formal analysis, D.S. (Dimitra Stavroulaki), V.A., I.K., D.S. (Dimitrios Skourtis), P.G.F., A.L., S.S., G.P. and D.K.; supervision, H.I., K.D. and D.M.H.; writing—review and editing, H.I., P.G.F., K.D. and D.M.H.; resources, H.I., K.D. and D.M.H.; data curation, H.I., K.D. and D.M.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research project was supported by the Hellenic Foundation for Research and Innovation (H.F.R.I.) under the "2nd Call for H.F.R.I. Research Projects to support Faculty Members & Researchers" (Project Number: 2762).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** None of the authors have any conflicts of interest including any financial, personal or other relationships with other people or organizations.

### **Abbreviations**

BOC, *tert*-butyloxycarbonyl; CD, circular dichroism; DCl, deuterium chloride; DMF, dimethylformamide; DMSO, methyl sulfoxide; DOX, doxorubicin; DLS, dynamic light scattering; DTT, dithiothreitol; EE, encapsulation efficiency; FBS, fetal bovine serum; FT-IR, Fourier transform infrared spectroscopy; GSH, glutathione; HVL, high vacuum line; LC, loading capacity; MMP2, matrix metalloproteinase 2; MSNs, mesoporous silica nanoparticles; MWCO, molecular weight-cutoff; MilliQ®, water purification systems from Merck Millipore; NCAs, *N*-carboxy anhydrides; NMR, nuclear magnetic resonance; NPs, nanoparticles; PBS, phosphate buffered saline; P(Cys), poly(L-cysteine); PEO, poly(ethylene oxide); P(His), poly(L-histidine); PPhe, poly(L-phenylalanine); PTFE, poly(tetrafluoroethylene); Rg, radius of gyration; Rh, hydrodynamic radius; ROP, ring-opening polymerization; ROS, reactive oxidative species; SCL, shell cross-linked; SEC, size exclusion chromatography; SLS, static light scattering; SRB, sulforhodamine B; TCA, trichloroacetic acid; TEA, triethylamine; TEM, transmission electron microscopy; TFA, trifluoroacetic acid; THF, tetrahydrofuran.
