*3.4. Quantification of Her2 on Cells*

PC-3 cells were found to express 12 <sup>×</sup> <sup>10</sup><sup>3</sup> <sup>±</sup> 0.5 <sup>×</sup> <sup>10</sup><sup>3</sup> Her2 receptors per cell. This was next compared to the expression of Her2 receptor on breast cancer cells MDA-MB-231, MDA-MB-453 and SKBR3 (reference cells for Her2 expression), as shown in Figure *Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 4. 8 of 16 *Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 8 of 16

**Figure 4.** Comparison of Her2 receptors on cell surface for MDA-MB-231, PC-3, MDA-MB-453 and SKBR3. **Figure 4.** Comparison of Her2 receptors on cell surface for MDA-MB-231, PC-3, MDA-MB-453 and SKBR3. **Figure 4.** Comparison of Her2 receptors on cell surface for MDA-MB-231, PC-3, MDA-MB-453 and SKBR3.

#### *3.5. 3D In Vitro 3.5. 3D In Vitro 3.5. 3D In Vitro*

#### 3.5.1. Lipidic Antiproliferative Activity 3.5.1. Lipidic Antiproliferative Activity 3.5.1. Lipidic Antiproliferative Activity

For both liposomes and immunoliposomes and for both formulations, the nontoxic concentration in empty liposomes was observed for lipid concentrations <10 nM for PC-3 cells treated at days 1 and 8 with viability determined in bioluminescence at day 15 (Figure 5). These results agree with those obtained in 2D (data not shown). For both liposomes and immunoliposomes and for both formulations, the nontoxic concentration in empty liposomes was observed for lipid concentrations <10 nM for PC-3 cells treated at days 1 and 8 with viability determined in bioluminescence at day 15 (Figure 5). These results agree with those obtained in 2D (data not shown). For both liposomes and immunoliposomes and for both formulations, the nontoxic concentration in empty liposomes was observed for lipid concentrations <10 nM for PC-3 cells treated at days 1 and 8 with viability determined in bioluminescence at day 15 (Figure 5). These results agree with those obtained in 2D (data not shown).

**Figure 5.** Cell viability (%) of PC-3 when exposed to Empty-Li-2. **Figure 5.** Cell viability (%) of PC-3 when exposed to Empty-Li-2. **Figure 5.** Cell viability (%) of PC-3 when exposed to Empty-Li-2.

Following these results, we decided to work with formulation 2 at two nontoxic lipid concentrations (2 and 8 nM) and an encapsulated ASO at 150 nM. Encapsulation rate for each batch Following these results, we decided to work with formulation 2 at two nontoxic lipid concentrations (2 and 8 nM) and an encapsulated ASO at 150 nM. Encapsulation rate for each batch Following these results, we decided to work with formulation 2 at two nontoxic lipid concentrations (2 and 8 nM) and an encapsulated ASO at 150 nM. Encapsulation rate for each batch was 41 ± 6%.

#### was 41 ± 6%. was 41 ± 6%. 3.5.2. Liposomal Antiproliferative Activity

summarized in Figure 6 and Figure 7.

summarized in Figure 6 and Figure 7.

3.5.2. Liposomal Antiproliferative Activity All the measures were performed in triplicate; these protocols (protocols 1 and 2) are 3.5.2. Liposomal Antiproliferative Activity All the measures were performed in triplicate; these protocols (protocols 1 and 2) are All the measures were performed in triplicate; these protocols (protocols 1 and 2) are summarized in Figures 6 and 7.

*Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 9 of 16

**Figure 6.** Protocol 1: liposomal antiproliferative activity with short exposition time. 3D in vitro assessment with spheroid treatment protocol 1. All the measures were performed in triplicate. **Figure 6.** Protocol 1: liposomal antiproliferative activity with short exposition time. 3D in vitro assessment with spheroid treatment protocol 1. All the measures were performed in triplicate. **Figure 6.** Protocol 1: liposomal antiproliferative activity with short exposition time. 3D in vitro assessment with spheroid treatment protocol 1. All the measures were performed in triplicate.

**Figure 7.** Protocol 2: liposomal antiproliferative activity with long exposition time. 3D in vitro **Figure 7.** Protocol 2: liposomal antiproliferative activity with long exposition time. 3D in vitro assessment with spheroid treatment protocol 2. All the measures were performed in triplicate. **Figure 7.** Protocol 2: liposomal antiproliferative activity with long exposition time. 3D in vitro assessment with spheroid treatment protocol 2. All the measures were performed in triplicate.

assessment with spheroid treatment protocol 2. All the measures were performed in triplicate.

ASO-iLi-2 at 8 nM with both schedules.

Empty liposomes and immunoliposomes showed no in vitro cytotoxicity (data not shown). Results of cytotoxic studies with protocol 1 are summarized in Figure 8. Greater cytotoxicity was observed in PCa exposed to liposomes with both treatment schemes. No effect was detected with Empty liposomes and immunoliposomes showed no in vitro cytotoxicity (data not shown). Results of cytotoxic studies with protocol 1 are summarized in Figure 8. Greater cytotoxicity was observed in PCa exposed to liposomes with both treatment schemes. No effect was detected with ASO-iLi-2 at 8 nM with both schedules. Empty liposomes and immunoliposomes showed no in vitro cytotoxicity (data not shown). Results of cytotoxic studies with protocol 1 are summarized in Figure 8. Greater cytotoxicity was observed in PCa exposed to liposomes with both treatment schemes. No effect was detected with ASO-iLi-2 at 8 nM with both schedules.

*Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 10 of 16

*Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 10 of 16

**Figure 8.** Cell viability (%) of 5000 PC-3 cell spheroids when exposed to ASO-Li-2 at 8 nM and ASOiLi-2 at 8 nM for 4h at day 3 and days 3–10 (\*\*\*, 0; \*\*, 0.001; \* 0.01). **Figure 8.** Cell viability (%) of 5000 PC-3 cell spheroids when exposed to ASO-Li-2 at 8 nM and ASO-iLi-2 at 8 nM for 4h at day 3 and days 3–10 (\*\*\*, 0; \*\*, 0.001; \* 0.01). **Figure 8.** Cell viability (%) of 5000 PC-3 cell spheroids when exposed to ASO-Li-2 at 8 nM and ASOiLi-2 at 8 nM for 4h at day 3 and days 3–10 (\*\*\*, 0; \*\*, 0.001; \* 0.01).

Results of cytotoxic studies with protocol 2 at low lipid concentration are summarized in Figure 9. Greater cytotoxicity was observed in PCa exposed to immunoliposomes encapsulated with 150 nM of ASO and to lipid concentration of 2 nM (*p* = 0.039). No cytotoxic effect on PCa cells was detected after ASO-Li-2 treatment at low lipid concentration. Results of cytotoxic studies with protocol 2 at low lipid concentration are summarized in Figure 9. Greater cytotoxicity was observed in PCa exposed to immunoliposomes encapsulated with 150 nM of ASO and to lipid concentration of 2 nM (*p* = 0.039). No cytotoxic effect on PCa cells was detected after ASO-Li-2 treatment at low lipid concentration. Results of cytotoxic studies with protocol 2 at low lipid concentration are summarized in Figure 9. Greater cytotoxicity was observed in PCa exposed to immunoliposomes encapsulated with 150 nM of ASO and to lipid concentration of 2 nM (*p* = 0.039). No cytotoxic effect on PCa cells was detected after ASO-Li-2 treatment at low lipid concentration.

**Figure 9.** Cell viability (%) of 5000 PC-3 cell spheroids when exposed to Empty-iLi-2 at 2 nM, ASO-Li-2 at 2 nM and ASO-iLi-2 at 2 nM (\*\*\*, 0; \*\*, 0.001; \* 0.01). **Figure 9.** Cell viability (%) of 5000 PC-3 cell spheroids when exposed to Empty-iLi-2 at 2 nM, ASO-Li-2 at 2 nM and ASO-iLi-2 at 2 nM (\*\*\*, 0; \*\*, 0.001; \* 0.01). **Figure 9.** Cell viability (%) of 5000 PC-3 cell spheroids when exposed to Empty-iLi-2 at 2 nM, ASO-Li-2 at 2 nM and ASO-iLi-2 at 2 nM (\*\*\*, 0; \*\*, 0.001; \* 0.01).

Results of cytotoxic studies with protocol 2 at high lipid concentration are summarized in Figure 10. A greater cytotoxicity was observed in PCa exposed to immunoliposomes encapsulated 150 nM of ASO (*p* = 0.0041 vs. Empty-iL-2 and *p* = 0.0039 vs. ASO-Li-2). Results of cytotoxic studies with protocol 2 at high lipid concentration are summarized in Figure 10. A greater cytotoxicity was observed in PCa exposed to immunoliposomes encapsulated 150 nM of ASO (*p* = 0.0041 vs. Empty-iL-2 and *p* = 0.0039 vs. ASO-Li-2). Results of cytotoxic studies with protocol 2 at high lipid concentration are summarized in Figure 10. A greater cytotoxicity was observed in PCa exposed to immunoliposomes encapsulated 150 nM of ASO (*p* = 0.0041 vs. Empty-iL-2 and *p* = 0.0039 vs. ASO-Li-2).

*Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 11 of 16

*Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 11 of 16

**Figure 10.** Cell viability (%) of 5000 PC-3 cell spheroids when exposed to Empty-iLi-2 at 8 nM, ASO-Li-2 at 8 nM and ASO-iLi-2 at 8 nM (\*\*\*, 0; \*\*, 0.001; \* 0.01). **Figure 10.** Cell viability (%) of 5000 PC-3 cell spheroids when exposed to Empty-iLi-2 at 8 nM, ASO-Li-2 at 8 nM and ASO-iLi-2 at 8 nM (\*\*\*, 0; \*\*, 0.001; \* 0.01). **Figure 10.** Cell viability (%) of 5000 PC-3 cell spheroids when exposed to Empty-iLi-2 at 8 nM, ASO-Li-2 at 8 nM and ASO-iLi-2 at 8 nM (\*\*\*, 0; \*\*, 0.001; \* 0.01).

Cell viability results obtained were confirmed by microscopic observation (Figure 11). Observations were made on days 1 (24 h after cell seeding), 8 and 15. Cell viability results obtained were confirmed by microscopic observation (Figure 11). Observations were made on days 1 (24 h after cell seeding), 8 and 15. Cell viability results obtained were confirmed by microscopic observation (Figure 11). Observations were made on days 1 (24 h after cell seeding), 8 and 15.

**Figure 11.** Spheroid (5000 cells) microscopy observations. **Figure 11.** Spheroid (5000 cells) microscopy observations. **Figure 11.** Spheroid (5000 cells) microscopy observations.

Microscopy observations were consistent with bioluminescence ones. On day 15, spheroids treated with ASO-iLi-2 at 8 nM seemed to present more cellular death than control and other treatment conditions. Microscopy observations were consistent with bioluminescence ones. On day 15, spheroids treated with ASO-iLi-2 at 8 nM seemed to present more cellular death than control and other treatment conditions. Microscopy observations were consistent with bioluminescence ones. On day 15, spheroids treated with ASO-iLi-2 at 8 nM seemed to present more cellular death than control and other treatment conditions.

#### **4. Discussion 4. Discussion 4. Discussion**

ASOs are a promising approach in oncology, and more particularly in CRPCa with the development of ASO targeting TCTP. ASOs are a promising approach in oncology, and more particularly in CRPCa with the development of ASO targeting TCTP. ASOs are a promising approach in oncology, and more particularly in CRPCa with the development of ASO targeting TCTP.

While ASO has shown its efficacy in vitro, cellular uptake has proven to be much more challenging. In this respect, we have here studied a new encapsulated formulation of ASO, designed to penetrate in cells. In this work, we have developed innovative second- and third-generation liposomal formulations encapsulating ASO, so as to enhance cellular penetration and effectiveness While ASO has shown its efficacy in vitro, cellular uptake has proven to be much more challenging. In this respect, we have here studied a new encapsulated formulation of ASO, designed to penetrate in cells. In this work, we have developed innovative second- and third-generation liposomal formulations encapsulating ASO, so as to enhance cellular penetration and effectiveness While ASO has shown its efficacy in vitro, cellular uptake has proven to be much more challenging. In this respect, we have here studied a new encapsulated formulation of ASO, designed to penetrate in cells. In this work, we have developed innovative second- and third-generation liposomal formulations encapsulating ASO, so as to enhance cellular penetration and effectiveness in PCa cells [33–36].

in PCa cells [33–36]. Pegylated immunoliposomes, thanks to their stealthiness and size compatible with the EPR effect, provide a unique opportunity for both passive and active targeting of tumors. To this end, liposomal formulations must take into account two important parameters: the nature of the ASO (i.e., in PCa cells [33–36]. Pegylated immunoliposomes, thanks to their stealthiness and size compatible with the EPR effect, provide a unique opportunity for both passive and active targeting of tumors. To this end, liposomal formulations must take into account two important parameters: the nature of the ASO (i.e., Pegylated immunoliposomes, thanks to their stealthiness and size compatible with the EPR effect, provide a unique opportunity for both passive and active targeting of tumors. To this end, liposomal formulations must take into account two important parameters: the nature of the ASO (i.e., its hydrophilic nature and its anionic character) and the necessity of a diameter below 200 nm

its hydrophilic nature and its anionic character) and the necessity of a diameter below 200 nm for

its hydrophilic nature and its anionic character) and the necessity of a diameter below 200 nm for

for EPR effect. Here, anti-Her2 trastuzumab was used as a targeting agent because Her2 is frequently expressed in several cancers such as breast cancer or gastric/gastroesophageal cancers [37].

We found that the level of Her2 expression in PC-3 was relatively low, so previous work on MDA-MB231 cells with even a lower level of target expression than PC-3 showed promising results in our laboratory. Still, the rationale for targeting Her2 on PC-3 cells could be questioned here. Because the PC-3 cells are the cells mostly used as an experimental model for CRPa [38,39], they were considered as a fully suitable model for evaluating the usefulness of transporting ASOs with immunoliposomes in prostate cancer, especially because we have demonstrated previously that higher efficacy with trastuzumab-grafted liposomes was not necessarily correlated to a high level of Her2 expression.

The innovative nature of this approach consisted in the combination of the advantages of a small diameter (i.e., <150 nm) of lipid–nucleic acid nanoparticles, improving ASO drug delivery through passive targeting, with the advantages of active targeting (i.e., anti-Her2) [40–42]. Prostate tumors are highly vascularized tumors, thus making this tumor eligible for EPR effects [43,44].

Previous studies have highlighted the complexity of defining optimal formulations yielding acceptable encapsulation rates with other hydrophilic active agents such as siRNA [45]. In our study, the difficulty was to combine acceptable encapsulation rates with small-sized nanoparticles while allowing trastuzumab engraftment.

In the present study, we showed that is it possible to design ASO liposomes and immunoliposomes. We have developed two different liposomal formulations showing good performances in terms of size, encapsulation rate and stability. DOTAP is a cationic lipid used to optimize liposome stability and increase anionic oligonucleotide ASO encapsulation rate. Chol and PC are neutral lipids frequently used for liposome formulation. The two tested formulations differ by PC presence in order to increase liposome stability. Finally, Mal-PEG is a common stealth agent used for all kinds of nanoparticles [46,47].

We worked with two nontoxic lipid concentrations (2 nM and 8 nM) and an encapsulated ASO at 150 nM. Indeed, lipid concentration plays a major role in liposome stability and leakage from the membrane. Low cholesterol concentration decreases stability and increases drug release [48]. Testing two lipid concentration levels allowed differences in cytotoxic effect to be highlighted.

Moreover, cholesterol affects the plasticity of the liposomal membrane; i.e., increasing cholesterol decreases the plasticity and increases the rigidity of the membranes [49]. Cholesterol was therefore a critical component because, for hydrophilic components such as ASO, membrane rigidity determines the release rate of the content [50].

A significant difference was found between formulations using fast agitation and the two other formulations (*p* < 0.001). This difference can be explained by destabilization and weakening of liposomes during fast agitation.

Following these results, we decided to work with formulation 2 composed of DOTAP/Mal-PEG/PC/Chol in the molar ratio 20:20:58:2. The encapsulation rate for each batch was 41 ± 6%.

Liposomes and immunoliposomes were compared in terms of physical characteristics, size suitable for EPR effect and encapsulation rates. The extraction of the ASO was optimized using a chloroform–methanol mixture. Finally, soft agitation with thin-film method was selected as the best encapsulation method, yielding encapsulation rates of about 50%, i.e., twice as much as the other methods we tested.

However, for the present proof-of-concept study, aiming at demonstrating that encapsulating ASOs in lipidic carriers helps to increase their efficacy, we decided to work on freshly prepared batches, rather than trying to improve shelf-stability.

Antiproliferative assays were performed on PCa-3 cells using both 2D and 3D models. Spheroids (i.e., 3D) are considered as a better model for evaluating the efficacy of nanoparticles [51]; however, 2D models were useful for determining IC50s of free ASO and free trastuzumab, as well as for determining the nontoxic concentrations of blank liposomes. These preliminary results on 2D models helped us to determine the concentrations (i.e., 2 and 8 nM) to be used for the 3D testing without confounding factors such as the direct toxic effect of overly concentrated lipids.

When using 3D models, the antiproliferative activity of free ASO was low and fully in line with that already described in the literature, thus confirming the need to provide ASO with a carrier to further increase its efficacy. Empty liposomes and blank immunoliposomes did not show antiproliferative activity. In 3D culture, the nontoxic concentration in lipids was confirmed to be 10 nM.

For free drugs (i.e., free ASO and free trastuzumab), no efficacy was observed in 3D models (data not shown). These data confirmed that ASO needs to be encapsulated to be efficient. We subsequently developed two processing protocols taking into account the preliminary data available [22].

Regardless of the short exposition time (4 h) and delayed exposure (i.e., day 3 and days 3 and 10), we observed a significant superiority in the efficacy of the liposomes as compared to the immunoliposomes. On the other hand, in these conditions of brief exposure, the treatment of PCa cells by immunoliposomes did not show any efficacy.

This scheduling was previously used in 2D models with PC-3 and already showed an effect in downregulation of TCTP expression and cell viability when using lipid-conjugated ASO carrier [12]. However, with our liposomes and immunoliposomes, these results were not confirmed. This loss of efficacy for immunoliposomes encapsulating ASO could come from this incubation time being too short to allow proper interaction between trastuzumab and Her2 receptors on PC-3 cells, preventing the efficient intracellular release of ASO. Indeed, the presence of the grafted antibody in the bilayer of the nanoparticle causes steric hindrance and is probably accompanied by a longer cellular uptake, resulting in a delayed release of ASO at the cellular level and limiting its efficacy.

In protocol 2 with extended exposure time, no significant differences were found between liposome-encapsulated ASO and empty liposome at all lipid concentrations. We have demonstrated significantly superior efficacy of ASO-iLi-2 at any lipid concentration for long exposure times when compared to liposome treatment.

In addition, for long exposure times, we have demonstrated significantly superior efficacy of ASO-iLi-2 at 8 nM lipid concentration when compared to ASO-Li-2 treatment.

Trastuzumab, therefore, seems to play a critical role in the active targeting of PC-3 cells, despite their low expression level of Her2. Indeed, free trastuzumab showed no efficacy in 3D models. Thus, the greater antiproliferative efficacy achieved with immunoliposomes as compared with liposomes encapsulating ASO cannot be directly related to some kind of direct trastuzumab cytotoxicity. Instead, better targeting of cancer cells by passive and active targeting due to a better distribution of the immunoliposomes in the core of the spheroid could explain this increase in efficacy.

Moreover, the differences in efficacy observed between immunoliposomes used at 2 and 8 nM can be explained by differences in the membrane plasticity of the carriers. Indeed 2 nM immunoliposomes contain less cholesterol than 8 nM immunoliposomes, and consequently, the membrane is less rigid, resulting in a possible leaking process due to poor stability. Conversely, the lipid concentration of 8 nM immunoliposomes allows better protection and subsequently also better intracellular penetration of ASO into cancer cells.

The challenge of this work was to highlight the possibility of encapsulating the ASO into lipidic nanoparticles so as to increase its efficacy. For both the developed liposomes and immunoliposomes, we have demonstrated that lipidic nanoparticles are suitable for encapsulating ASOs in terms of size, short-term stability and encapsulation rates. Results showed that both liposomes and immunoliposomes performed better than free ASO, thus suggesting that encapsulating ASO could help to increase its cytotoxicity. Because nanoparticles do not aim at changing the pharmacology of a drug, but rather aim at affecting the delivery and pharmacokinetics, we hypothesize here that lipids containing ASO could help carry the payload inside tumors.

#### **5. Conclusions**

In this proof-of-concept study, we have demonstrated that it is possible to increase the efficacy of ASO in the canonical PC-3 model for prostate cancer by using lipid carriers. Interestingly, immunoliposomes targeting Her2 have presented the most promising efficacy on 3D spheroids,

provided that incubation time was long enough, despite a low expression of Her2 in PC-3 cells. Conversely, short exposure times led to higher efficacy of liposomes as compared with immunoliposomes. This difference could come from the steric hindrance of the immunoliposomes requiring a delay in the release of ASO, thus making it a more suitable candidate for long-exposure schedules. Overall, this work suggests that shifting from standard intratumoral administration of oligonucleotides to systemic administration is feasible, provided that a suitable vehicle is developed.

Poor stability of encapsulated ASO obliged us to use extemporaneously prepared batches; however, this weakness could be fixed in the future, i.e., by lyophilization of the nanoparticles so as to enhance shelf-stability.

**Author Contributions:** Conceptualization, G.S. and R.F.; resources, C.P. and A.R.; supervision, P.R.; visualization, J.C.; funding acquisition, S.G. All authors have read and agreed to the published version of the manuscript.

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

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

#### **References**


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