**Enhanced Antisense Oligonucleotide Delivery Using Cationic Liposomes Grafted with Trastuzumab: A Proof-of-Concept Study in Prostate Cancer**

**Guillaume Sicard <sup>1</sup> , Clément Paris <sup>2</sup> , Sarah Giacometti <sup>1</sup> , Anne Rodallec <sup>1</sup> , Joseph Ciccolini <sup>1</sup> , Palma Rocchi 2,\* and Raphaëlle Fanciullino <sup>1</sup>**


Received: 13 November 2020; Accepted: 27 November 2020; Published: 29 November 2020

**Abstract:** Prostate cancer (PCa) is the second most common cancer in men worldwide and the fifth leading cause of death by cancer. The overexpression of TCTP protein plays an important role in castration resistance. Over the last decade, antisense technology has emerged as a rising strategy in oncology. Using antisense oligonucleotide (ASO) to silence TCTP protein is a promising therapeutic option—however, the pharmacokinetics of ASO does not always meet the requirements of proper delivery to the tumor site. In this context, developing drug delivery systems is an attractive strategy for improving the efficacy of ASO directed against TCTP. The liposome should protect and deliver ASO at the intracellular level in order to be effective. In addition, because prostate cancer cells express Her2, using an anti-Her2 targeting antibody will increase the affinity of the liposome for the cell and optimize the intratumoral penetration of the ASO, thus improving efficacy. Here, we have designed and developed pegylated liposomes and Her2-targeting immunoliposomes. Mean diameter was below 200 nm, thus ensuring proper enhanced permeation and retention (EPR) effect. Encapsulation rate for ASO was about 40%. Using human PC-3 prostate cancer cells as a canonical model, free ASO and ASO encapsulated into either liposomes or anti-Her2 immunoliposomes were tested for efficacy in vitro using 2D and 3D spheroid models. While the encapsulated forms of ASO were always more effective than free ASO, we observed differences in efficacy of encapsulated ASO. For short exposure times (i.e., 4 h) ASO liposomes (ASO-Li) were more effective than ASO-immunoliposomes (ASO-iLi). Conversely, for longer exposure times, ASO-iLi performed better than ASO-Li. This pilot study demonstrates that it is possible to encapsulate ASO into liposomes and to yield antiproliferative efficacy against PCa. Importantly, despite mild Her2 expression in this PC-3 model, using a surface mAb as targeting agent provides further efficacy, especially when exposure is longer. Overall, the development of third-generation ASO-iLi should help to take advantage of the expression of Her2 by prostate cancer cells in order to allow greater specificity of action in vivo and thus a gain in efficacy.

**Keywords:** liposomes; immunoliposomes; antisense oligonucleotides; prostate cancer

### **1. Introduction**

Prostate cancer (PCa) is the second most common cancer in men worldwide with 1.3 million new cases in 2018. PCa is the fifth leading cause of death by cancer with more than 360,000 deaths in 2018, despite a decrease in its incidence by 6% over 2005–2009 [1,2]. At initial diagnosis, treatment depends on the stage based on Gleason score, the patient's characteristics and the PSA level [3,4]. Surgical treatment, i.e., prostatectomy, is the standard of care, but patients with advanced disease (i.e., stages III or IV, high Gleason score) are precluded. The first-line therapy for advanced PCa is castration therapy which consists in androgen deprivation since it is a hormone-sensitive cancer [5]. After a period of therapeutic response, usually 1–3 years, patients will ultimately become resistant to the therapy and develop metastases. A new approach is therefore needed for these castration-resistant PCa (CRPCa) patients.

The overexpression of the TCTP protein plays an important role in PCa and most particularly in CRPCa [6]. Indeed, this protein is involved in progression of the disease and therapeutic failure. The interactions between TCTP and p53 and their negative feedback regulation loop are responsible for progression and invasion of PCa [7,8]. Recently, antisense technology has emerged as a promising strategy in cancer [9]. The principle of this approach is the sequence-specific binding of an antisense oligonucleotide (ASO) to target mRNA, thus preventing gene translation [10]. The development of an ASO directed against TCTP seems therefore to be an interesting strategy [11]. The shutting down of TCTP by ASO is expected to restore apoptosis and sensitivity to hormone-therapy and chemotherapy of cancer cells. ASOs are only active after cell uptake, and therefore, a carrier is necessary to help them pass the membranes. In this context, developing carriers to transport ASO is an attractive strategy, especially since nanoparticles are increasingly considered to stretch the efficacy/toxicity balance of a variety of anticancer agents or payloads [12–14]. Various other technological approaches such as the direct pegylation of compounds [15] or using Nab conjugates [16] or antibody–drug conjugates [17,18] illustrate this major trend to develop drug carriers in oncology today. One of the critical points when developing nanoparticles is based on their size being <200 nm. Nanoparticles leave the vascular compartment and accumulate in the interstitial space next to the tumor. This phenomenon is called passive targeting or the enhanced permeability and retention (EPR) effect [19].

Our study is based upon this double trend of encapsulation and use of therapeutic monoclonal antibodies to improve the specificity of nanoparticles against tumors. The conjunction of these two concepts results in a new nanoparticle, the antibody nanoconjugate (ANC), more commonly called the immunoliposome [20,21]. In this study, we present the early development steps of an innovative stealth liposomal ASO nanoparticle targeting prostate cancer through anti-Her2 functionalization [22] (Figure 1).

*Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 3 of 16

**Figure 1.** Schematic representation of the immunoliposome structure and composition. **Figure 1.** Schematic representation of the immunoliposome structure and composition.

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

#### *2.1. Cell Lines 2.1. Cell Lines*

Experiments were carried on canonical human prostate cancer cell line PC-3 (American Type Culture Collection, Rockville, MD, USA) Cells were cultured in RPMI supplemented with 10% FBS, 1% penicillin and 0.16% kanamycin and grown in a humidified 5% CO2 incubator at 37 °C. Cells were regularly authenticated in terms of cell viability, morphology and doubling time. For Her2 characterization, breast cancer cell lines, i.e., MDA231, MDA 453 and SKBR3, were used (American Type Culture Collection, Manassas, VA, USA). Experiments were carried on canonical human prostate cancer cell line PC-3 (American Type Culture Collection, Rockville, MD, USA) Cells were cultured in RPMI supplemented with 10% FBS, 1% penicillin and 0.16% kanamycin and grown in a humidified 5% CO<sup>2</sup> incubator at 37 ◦C. Cells were regularly authenticated in terms of cell viability, morphology and doubling time. For Her2 characterization, breast cancer cell lines, i.e., MDA231, MDA 453 and SKBR3, were used (American Type Culture Collection, Manassas, VA, USA).

### *2.2. Drugs and Chemicals*

*2.2. Drugs and Chemicals*  1,2-distearoyl-*sn*-glycero-3-phosphoethanolamine-N-(aleimide(polyethyleneglycol)-2000) (Mal-PEG) and 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) were purchased from COGER (Paris, France). Egg yolk phosphatidylcholine (PC) and cholesterol (Chol) were purchased from Sigma (St-Quentin-Fallavier, France). ASO was purchased from Eurofins (Les Ullis, France). 2 iminothiolane (Traut's reagent) and Draq5 were purchased from Fisher Scientific (Illkirch-Graffenstaden, France). QuantiBRITE phycoerythrin (PE) and PE Mouse Anti-Human Her-2/neu were purchased from BD Biosciences (San Jose, CA, USA). Trastuzumab (Herceptin) was kindly given by Genentech (South San Francisco, CA, USA). All other reagents were of analytical grade. 1,2-distearoyl-*sn*-glycero-3-phosphoethanolamine-N-(aleimide(polyethyleneglycol)-2000) (Mal-PEG) and 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) were purchased from COGER (Paris, France). Egg yolk phosphatidylcholine (PC) and cholesterol (Chol) were purchased from Sigma (St-Quentin-Fallavier, France). ASO was purchased from Eurofins (Les Ullis, France). 2-iminothiolane (Traut's reagent) and Draq5 were purchased from Fisher Scientific (Illkirch-Graffenstaden, France). QuantiBRITE phycoerythrin (PE) and PE Mouse Anti-Human Her-2/neu were purchased from BD Biosciences (San Jose, CA, USA). Trastuzumab (Herceptin) was kindly given by Genentech (South San Francisco, CA, USA). All other reagents were of analytical grade.

#### *2.3. ASO Stability in Solvents*

was resuspended in water, and then 90 μL was injected.

*2.3. ASO Stability in Solvents*  ASO stability in three solvents, i.e., NaCl 0.9%, water and methanol, was tested over one month. HPLC detection was performed on an HPLC (Agilent 1260, Agilent, Les Ulis, France). The HPLC column Xbrige OST C18 2.5 μm 4.6 × 50 mm (Waters, Guyancourt, France) was equilibrated at a flow rate of 0.8 mL/min. Eluant A contained 0.1 M TEAA (Triethylammonium acetate) in 5% ACN (acetonitrile) in water and eluant B contained ACN. The elution gradient was 0 to 45% in 10 min. ASO ASO stability in three solvents, i.e., NaCl 0.9%, water and methanol, was tested over one month. HPLC detection was performed on an HPLC (Agilent 1260, Agilent, Les Ulis, France). The HPLC column Xbrige OST C18 2.5 µm 4.6 × 50 mm (Waters, Guyancourt, France) was equilibrated at a flow rate of 0.8 mL/min. Eluant A contained 0.1 M TEAA (Triethylammonium acetate) in 5% ACN (acetonitrile) in water and eluant B contained ACN. The elution gradient was 0 to 45% in 10 min. ASO was detected at the wavelength of 260 nm. Ninety microliters of ASO in NaCl 0.9% and ASO

was detected at the wavelength of 260 nm. Ninety microliters of ASO in NaCl 0.9% and ASO in water were directly injected. For ASO in MeOH, 100 μL of sample was evaporated to dryness; the sample in water were directly injected. For ASO in MeOH, 100 µL of sample was evaporated to dryness; the sample was resuspended in water, and then 90 µL was injected.

#### *2.4. Pegylated Liposome Preparation*

Two different compositions of liposomes were studied: formulation 1, using DOTAP, Mal-PEG and Chol, and formulation 2, using DOTAP, PC, Mal-PEG and Chol.

Both compositions were prepared using the classic thin-film method [23]. Briefly, lipids were dissolved in methanol as organic solvent. Methanol was then removed by rotary evaporation (Laborota 4003, Heidolph Instruments, Schwabach, Germany) at 38 ◦C under vacuum to avoid further toxicity. After 30 min, a thin lipid film was obtained. To remove the residual solvent, lipid film was dried under a stream of nitrogen for 2 h at room temperature. The film was then hydrated with a 5% *v*/*v* dextrose solution in water for formulation 1 or a 0.9% *v*/*v* sodium chloride solution in water for formulation 2, and then multilamellar vesicle (MLV) liposomes were obtained. Extrusion step was performed to reduce and homogenize liposomes in size through two 0.1 µm and two 0.08 µm polycarbonate pore membranes (Nucleopore, Whatman, Maidstone, UK) using LipoFast LF-50, and then small unilamellar vesicle (SUV) liposomes were obtained [24].

For each formulation, liposomes (i.e., ASO-Li-1 for formulation 1 and ASO-Li-1 for formulation 2) and immunoliposomes (i.e., ASO-iLi-1 and ASO-iLi-2) were generated.

#### *2.5. Pegylated Immunoliposome Preparation: Encapsulation Strategies*

Different encapsulation strategies were tested with both formulations.

With formulation 1, ASO was introducing in methanol with lipids during the thin lipid film formation. Extraction from liposome was performed by 100 µL methanol for 100 µL liposome.

With formulation 2, three different strategies were tested. First, ASO was included in aqueous solvent, i.e., sodium chloride 0.9%, during the hydration step of the lipidic film. ASO was also included in preformed liposomes by contact using other strategies, namely soft agitation by rotation at 38 ◦C or fast agitation using bar magnet at 38 ◦C.

Besides, a new extraction technique was used with a chloroform–methanol solution (1:2 ratio) [25]. For each 100 µL of liposome solution, we added 375 µL of chloroform–methanol solution. Then, 125 µL of chloroform was added to the sample and vortexed. Afterward, 125 µL of MilliQ water was added to the sample and vortexed again. Finally, the sample was centrifugated at 1500× *g* for 90 s. The topper phase contained the ASO freed from the lipids. The entire aqueous phase was recuperated, evaporated and reconstituted with 100 µL of MilliQ water.

#### *2.6. Pegylated Immunoliposome Preparation: Antibody Engraftment*

Trastuzumab engraftment was performed from previously obtained liposome, generating immunoliposome.

The engraftment was carried out by maleimide–thiol conjugation after having previously derivatized the trastuzumab with thiol function. To this end, trastuzumab was first dissolved in a 0.1 M sodium phosphate-buffered saline (PBS), pH 8.0, containing 5 mM ethylene diamine tetra-acetic acid, and mixed under constant shaking for 2 h at room temperature with a Traut's reagent solution at 1:10 molar ratio (Traut's/trastuzumab). Thiolated trastuzumab was then directly mixed with the pegylated liposomes at 1:127 molar ratio (trastuzumab/MAL-PEG).

The mixture was kept under constant shaking at 4 ◦C overnight. Unbound trastuzumab and free ASO were removed from the liposomal solution using 6000× *g* centrifugation on MWCO 300 KDa Vivaspin (VWR, Fontenay-sous-Bois, France) [26].

#### *2.7. Size and Polydispersity Study*

Size and polydispersity index (PDI) of liposomes and immunoliposomes were measured by dynamic light scattering (DLS). Analysis parameters were as follows: medium: PBS solution, viscosity of

water: 0.8937, temperature: 25 ◦C, dielectric constant: negative but not used in these measurements, nanoparticles: liposomes, refractive index of water: 1.333 cP, detection angle: 173◦ , wavelength: 632.8 nm, software for analysis of data: Zetasizer Nano software v3.30.

Liposomes and immunoliposomes were diluted in a PBS solution and then analyzed by a Zetasizer Nano S (Malvern Instruments, Malvern, UK). Liposomal preparations were considered unimodal for a PDI < 0.2. The measures were performed extemporaneously after liposome formation for both formulations in triplicate.

Stability study was performed at 7, 14 and 28 days after liposome formation.

#### *2.8. ASO Encapsulation Rate*

ASO encapsulation rate was only determined for formulation 2 by fluorescence (495/520 nm). After grafting ASO with FITC in 30 (Eurofins, Les Ullis, France), ASO–FITC encapsulation rate was determined by measuring the FITC fluorescence at 520 nm (PHERAstar FSX, BMG Labtech, Ortenberg, Germany) [27]. All the measures were performed in triplicate.

#### *2.9. Quantification of HER2 on Cells*

Flow cytometry analysis allowed measuring the expression of Her2 on the surface of PC-3 cells [28]. As previously described [29], QuantiBRITE PE (BD Biosciences, San Jose, USA) was used to estimate the absolute number of Her2 receptors on cell membranes. About 100,000 cells of PC-3 were incubated under saturated conditions with PE Mouse Anti-Human HER-2/neu (BD Biosciences, San Jose, USA) for 30 min at 4 ◦C before being rinsed with PBS. IgG2a-PE anti-mouse antibodies (Fisher Scientific, Illkirch, France) were used for isotopic control. Analysis was then immediately performed on Gallios Beckman Coulter. Assuming our anti-HER-2 PE antibody has a 1:1 fluorochrome/antibody ratio, PE median fluorescence intensity (MFI) was measured for all cell lines and reported on a log–log graph with MFI vs. PE molecules, after subtracting isotopic control MFI. All the measures were performed in triplicate.

#### *2.10. In Vitro Assays*

Spheroids were obtained with PC-3 cells seeded with 20% methylcellulose solution on a 96-well U-bottom plate for at least 24 h before the experiment began. Different drug concentrations and scheduling conditions were tested on 5000-cell spheroids. Viability was assessed by bioluminescence assay. The cell viability in bioluminescence was determined on PC-3 cells using luminescent cell viability assay (CellTiter-Glo, Promega, Madison, WI, USA) and bioluminescence reading (PHERAstar FSX; BMG Labtech, Ortenberg, Germany). Cellular uptake was observed using confocal microscopy (TCS SP2 Leica) coupled to a digital camera.

Using 5000-cell spheroids, we determined nontoxic concentrations of lipid treated on day 1 and day 8 with viability determined in bioluminescence at day 15.

Subsequently, in a first protocol, we tested one nontoxic concentration of lipid (i.e., 8 nM) following day 3 and day 10 (after spheroid formation) treatment schedule with viability assay on day 15 or only at day 3 with viability assay at day 15. Encapsulated ASO concentration (i.e., after lipidic film formation) at 150 nM was tested. Cells were exposed to treatment for four hours on Day 3 and/or day 10. After four hours of exposure, treatment was removed and replaced by supplemented RPMI.

In a second protocol, using 5000-cell spheroids, we tested two nontoxic concentrations of lipid (i.e., 2 and 8 nM) following day 3 and day 10 treatment schedule with viability assay on day 15. Encapsulated ASO concentration (i.e., after lipidic film formation) at 150 nM was tested. Empty liposomes and immunoliposomes, for each nontoxic lipid concentration, were used as control.

### *2.11. Statistical Analysis*

Formulation experiments were performed at least in triplicate and data were represented as mean ± SD or ±standard error of the mean. Statistical analyses were performed on MedCalc 17.2.1. Software (MedCalc, Acacialaan, Belgium). *Pharmaceutics* **2020**, *12*, x FOR PEER REVIEW 6 of 16

In vitro experiment was performed at least in triplicate and data were represented as mean ± SD or ±standard error of the mean. All statistical analyses were performed with car [30] and multcomp [31] packages of the software R [32]. In vitro experiment was performed at least in triplicate and data were represented as mean ± SD or ± standard error of the mean. All statistical analyses were performed with car [30] and multcomp [31] packages of the software R [32].

#### **3. Results 3. Results**

#### *3.1. ASO Stability in Solvents 3.1. ASO Stability in Solvents*

ASO stability has been studied in three different solvents. Sodium chloride 0.9% solution was tested because it was used during the hydration step of the lipidic film during liposome formation, water was used because lyophilized ASO was reconstituted with it and methanol was the organic solvent used for lipid dissolution before lipid film formation and for liposome extraction. ASO stability has been studied in three different solvents. Sodium chloride 0.9% solution was tested because it was used during the hydration step of the lipidic film during liposome formation, water was used because lyophilized ASO was reconstituted with it and methanol was the organic solvent used for lipid dissolution before lipid film formation and for liposome extraction.

Results show that ASO is stable in the three solvents over 32 days. Thus, formulation tests can be carried out by including ASO in the organic phase (methanol) or in the hydration solvent (NaCl 0.9%) without fear of deterioration of the ASO (Figure 2). Results show that ASO is stable in the three solvents over 32 days. Thus, formulation tests can be carried out by including ASO in the organic phase (methanol) or in the hydration solvent (NaCl 0.9%) without fear of deterioration of the ASO (Figure 2).

**Figure 2.** Normalized percentage of full antisense oligonucleotide (ASO) length over time for three solvents. **Figure 2.** Normalized percentage of full antisense oligonucleotide (ASO) length over time for three solvents.

#### *3.2. Size and Polydispersity 3.2. Size and Polydispersity*

immunoliposomes.

Size and PDI are summarized in Table 1.

We have developed two lipidic compositions: DOTAP/Mal-PEG/Chol (29:2:69) and DOTAP/Mal-PEG/PC/Chol (20:20:58:2) respectively for ASO-Li-1/ASO-iLi-1 and ASO-Li-2/ASO-iLi-2. We have developed two lipidic compositions: DOTAP/Mal-PEG/Chol (29:2:69) and DOTAP/Mal-PEG/PC/Chol (20:20:58:2) respectively for ASO-Li-1/ASO-iLi-1 and ASO-Li-2/ASO-iLi-2. Size and PDI are summarized in Table 1.

**Table 1.** Size and polydispersity index (PDI) comparison for both formulations of liposomes and

**ASO-Li-2** 145.6 ± 4.1 0.080 ± 0.01

**Formulation Size (nm) ± SD PDI ± SD** 


**Table 1.** Size and polydispersity index (PDI) comparison for both formulations of liposomes and immunoliposomes.

We observed a statistically significant difference in size between ASO-Li-1 and ASO-iLi-1 (*p* = 0.016) and ASO-Li-2 and ASO-iLi-2 (*p* = 0.032, Student's *t*-test). We observed a statistically significant difference in size between ASO-Li-1 and ASO-iLi-1 (*p* = 0.016) and ASO-Li-2 and ASO-iLi-2 (*p* = 0.032, Student's *t*-test).

Stability study was performed on days 7, 14 and 28 after liposome formation. For formulation 1, size showed an increase both for ASO-Li-1 (*p* = 0.007, Student's *t*-test) and ASO-iLi-1 (*p* = 0.002, Student's *t*-test). Given these results, stability study was not performed on days 14 and 28. Stability study was performed on days 7, 14 and 28 after liposome formation. For formulation 1, size showed an increase both for ASO-Li-1 (*p* = 0.007, Student's *t*-test) and ASO-iLi-1 (*p* = 0.002, Student's *t*-test). Given these results, stability study was not performed on days 14 and 28.

For formulation 2, size showed no difference from first measures (*p* = 0.838 for ASO-Li-2 and *p* = 0.838 for ASO-iLi-2, one-way ANOVA testing). Results for size and PDI monitoring over time are summarized in Table 2. For formulation 2, size showed no difference from first measures (*p* = 0.838 for ASO-Li-2 and *p* = 0.838 for ASO-iLi-2, one-way ANOVA testing). Results for size and PDI monitoring over time are summarized in Table 2.

**Table 2.** Size and PDI comparison over time for formulation 2. **Table 2.** Size and PDI comparison over time for formulation 2.


#### *3.3. Pegylated Immunoliposome Preparation: Encapsulation Strategies 3.3. Pegylated Immunoliposome Preparation: Encapsulation Strategies*

*3.4. Quantification of Her2 on Cells* 

ASO–FITC was encapsulated into liposomes composed of DOTAP/Mal-PEG/PC/Chol in the molar ratio 20:20:58:2. Mean encapsulation rates of ASO–FITC were 21.14 ± 7.65% for fast agitation, 42.88 ± 3.80% for soft agitation and 39.00 ± 2.16% combined hydration solvent and soft agitation (Figure 3). No significant difference appears between the two formulations using soft agitation *p* = 0.484). Whether ASO was introduced in organic or in hydrophilic phase had no influence on ASO encapsulation. ASO–FITC was encapsulated into liposomes composed of DOTAP/Mal-PEG/PC/Chol in the molar ratio 20:20:58:2. Mean encapsulation rates of ASO–FITC were 21.14 ± 7.65% for fast agitation, 42.88 ± 3.80% for soft agitation and 39.00 ± 2.16% combined hydration solvent and soft agitation (Figure 3). No significant difference appears between the two formulations using soft agitation *p* = 0.484). Whether ASO was introduced in organic or in hydrophilic phase had no influence on ASO encapsulation.

**Figure 3.** Influence of different strategies on encapsulation rate (\*\*\*, 0; \*\*, 0.001; \* 0.01). **Figure 3.** Influence of different strategies on encapsulation rate (\*\*\*, 0; \*\*, 0.001; \* 0.01).

PC-3 cells were found to express 12 × 103 ± 0.5 × 103 Her2 receptors per cell. This was next
