*3.2.3 Novel approaches drug delivery platform with the integration of different factors*

In some cases, to ameliorate the level of treatment dual drug delivery suggests. For example, for carrying quercetin and gefitinib individually and together to PA-1 OC cells polyvinylpyrrolidone (PVP)-functionalized graphene oxide NPs (GO-PVP-NPs) as a system delivery was used. The results indicated combination delivery is more effective than individually in PA-1 cells [41].

### **Figure 3.**

*Tariquidar and doxorubicin conjugated with pH sensitive liposomes to overcome multidrug resistance of OC cells. This figure was obtained with permission from [37] under the terms of creative commons CC BY license.*

**181**

growth [43].

**Figure 4.**

*CC BY license.*

NP in tumor tissues [44].

*Nano Technology and Gas Plasma as Novel Therapeutic Strategies for Ovarian Cancer…*

Polymeric NPs and lipid-based NPs are the most usable nanocarriers that sometimes a combination of them make NPs suitable for drug delivery with high efficacy. In this experiment, a Pluronic F127 and a lipid-PEG stabilizer were used to generate NPs which have internal cubic phases are called cubosomes (CB) and external sponge phase. These NPs are conjugated with PTX against HEY cells and disrupt EGFR that overexpress in OC. Decreasing the cell viability and inhibiting the tumor

*Polymersomal doxorubicin with GE11peptide designed as an alternative to Clinical Liposomal Formulation for ovarian oncotherapy. This figure was obtained with permission from [40] under the terms of creative commons* 

Platinum-resistant OC (PROC) isn't possible to treat by conventional therapy for solving this problem, PROC is treated by CIS and wortmannin (Wtmn) as a DNA repair inhibitor conjugated with PEG-PLGA NPs. After treated A2780 cells, γH2AX foci as a DNA double-strand breaks marker analyzed for Wtmn activity and cytotoxicity. High solubility and stability are other properties of this dual nanocarrier. In vivo studies displayed the low concentration of drugs could inhibit tumor

Nucleic acid-based NPs is another nanovehicle for transferring drugs to OC cells. For instance, an annexin A2 aptamer (ndo28) bind to pRNA-3WJ NPs and design a GC rich sequence in NPs for linking DOX. Treating SKOV3 cells by this NP increase cytotoxicity, and xenograft mice models showed targeting and accumulation of this

*DOI: http://dx.doi.org/10.5772/intechopen.96387*

growth are the results of this study [42].

*Nano Technology and Gas Plasma as Novel Therapeutic Strategies for Ovarian Cancer… DOI: http://dx.doi.org/10.5772/intechopen.96387*

### **Figure 4.**

*Ovarian Cancer - Updates in Tumour Biology and Therapeutics*

pHSL/TQR/DOX (**Figure 3**) [37].

the tumor growth [39].

(**Figure 4**) [40].

*factors*

resistance. pHSL made from CHEMS (cholesteryl hemisuccinate), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and PEGylated lipid which DOX and TQR placed in the water and lipid phases respectively and this nano vehicle prolong circulation. Cytotoxicity was investigated by treatment OVCAR8/ADR cells with

Combination of FA ligand for specific target delivery and pH-sensitive NPs proposed phenomenal nanocarrier for ovarian oncotherapy. Magnetic NPs (MNPs) and MTX through carboxylic acid groups and amino groups of chitosan linked to chitosan copolymer and prepared thermos and pH-sensitive MTX-CSC@MNPs that conjugate with erlotinib (ETB) for target delivery. Since MTX and FA are similar structurally this nano vehicle absorbed with folate receptor on OVCAR-3 cells and prompt cytotoxicity and apoptosis induced by ETB [38]. Moreover, pH-sensitive Glucose/gluconic acid-coated magnetic NPs that linked to FA in the surface, enclosed DOX. External magnetic fields improve drug release in tumor tissue. For evaluating cell viability A2780, OVCAR3 and SKOV3 cells treated by these NPs and results demonstrated an increase in internalization and cytotoxicity. Analyzing the tissues of the SKOV3-Luc cell-xenografted nude mouse model showed accumulation of the drug in tumor cells more than other parts of the body that it causes to block

Drug released is also controlled by intracellular GSH concentration. GSH sensitive polymersomal DOX nano vehicle that modified by GE11 peptide (GE11- PS-Dox) is one of these NPs produced for treatment SKOV3 cells with a high level of epidermal growth factor receptor (EGFR). After drug delivery to tumor tissue and cancer cells, DOX enters the cell nucleus and inhibits tumor progress and increases cytotoxicity. The efficiency of this treatment is more than Lipo-Dox or Dox alone

*3.2.3 Novel approaches drug delivery platform with the integration of different* 

delivery is more effective than individually in PA-1 cells [41].

In some cases, to ameliorate the level of treatment dual drug delivery suggests. For example, for carrying quercetin and gefitinib individually and together to PA-1 OC cells polyvinylpyrrolidone (PVP)-functionalized graphene oxide NPs (GO-PVP-NPs) as a system delivery was used. The results indicated combination

*Tariquidar and doxorubicin conjugated with pH sensitive liposomes to overcome multidrug resistance of OC cells. This figure was obtained with permission from [37] under the terms of creative commons CC BY license.*

**180**

**Figure 3.**

*Polymersomal doxorubicin with GE11peptide designed as an alternative to Clinical Liposomal Formulation for ovarian oncotherapy. This figure was obtained with permission from [40] under the terms of creative commons CC BY license.*

Polymeric NPs and lipid-based NPs are the most usable nanocarriers that sometimes a combination of them make NPs suitable for drug delivery with high efficacy. In this experiment, a Pluronic F127 and a lipid-PEG stabilizer were used to generate NPs which have internal cubic phases are called cubosomes (CB) and external sponge phase. These NPs are conjugated with PTX against HEY cells and disrupt EGFR that overexpress in OC. Decreasing the cell viability and inhibiting the tumor growth are the results of this study [42].

Platinum-resistant OC (PROC) isn't possible to treat by conventional therapy for solving this problem, PROC is treated by CIS and wortmannin (Wtmn) as a DNA repair inhibitor conjugated with PEG-PLGA NPs. After treated A2780 cells, γH2AX foci as a DNA double-strand breaks marker analyzed for Wtmn activity and cytotoxicity. High solubility and stability are other properties of this dual nanocarrier. In vivo studies displayed the low concentration of drugs could inhibit tumor growth [43].

Nucleic acid-based NPs is another nanovehicle for transferring drugs to OC cells. For instance, an annexin A2 aptamer (ndo28) bind to pRNA-3WJ NPs and design a GC rich sequence in NPs for linking DOX. Treating SKOV3 cells by this NP increase cytotoxicity, and xenograft mice models showed targeting and accumulation of this NP in tumor tissues [44].

NP-drug conjugates (NDCs) are more effective than antibody-drug conjugates (ADCs) for loading monomethyl auristatin E (MMAE) in OC therapy. So, the results were very promising in the level of in vitro and in vivo, and inhibition of tumor growth and cytotoxicity in a patient-derived xenograft model of platinumresistant OC is twice as much in comparison with CIS administration [45].

Depolarization of mitochondria and augment the level of ROS that cause apoptosis and finally, cytotoxicity in tumor cells are other results of administration NPs individually or in combination with chemotherapy drugs. So, Gold NPs encapsulate theaflavin (tea-extracted polyphenols) (AuNP@TfQ ) as an apoptosis inducer in tumor cells. Anti-cancer activity of AuNP@TfQ enhanced by pristine theaflavin oxidation to its quinone derivative on the surface of gold NPs. The entrance of AuNP@TfQ into the PA-1 cells takes place through endocytosis. In this study caspase-3, Bax, Bad, BID, and BIM as pro-apoptotic markers and Bcl-2 and Bcl-was anti-apoptotic markers were evaluated [46].

But NPs individually can lead to cytotoxicity and apoptosis in tumor cells too. Gurunathan and colleagues proposed Ag NPs and ZnO NPs with a broad range of application in biomedical was used to treat OC individually and synergy with gemcitabine (GEM). Results represented a reduction in cell viability in a dose and time dependent manner and DNA double-strand break due to the overproduction of ROS and mitochondria dysfunction in both experiments [47, 48].

### **3.3 Gene delivery**

Herein, we focused on some gene delivery based examples used for OC treatment. NPs due to their properties are used as a vehicle for delivery of different nucleic acids such as siRNA, miRNA and shRNA. Increasing cytotoxicity by inducing apoptosis and suppressing tumor growth and volume are the common results obtain via silencing or downregulating oncogenes. Modifying NPs with ligands for improving delivery is also utilized in this technique.

### *3.3.1 NPs for siRNA delivery*

Using siRNA in oncotherapy because of low toxicity, high effectiveness and specificity is a terrific choice. To improve the efficiency and overcome the problem such as degradation by RNase and lack of the ability to penetrate the membrane cells, is better not to use naked siRNA. To facilitate the capability of siRNA and presence in proper concentration for silencing or downregulating of oncogenes, gene delivery systems play an essential role. Polymeric and lipid-based NPs are the most usable carriers for siRNA. Polymeric NPs especially PEI, PEG and chitosan with a positive charge easily bind to oligonucleotides with a negative charge through electrostatic interactions. While lipid NPs such as liposomes encapsulate siRNA in its aqueous core [30]. Polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol) modified FA (hyPEI-g-PCL-b-PEG-FA) is one of the examples polymeric NPs was used for transfer siRNA to SKOV-3/LUC cells with a high level of FRα [49].

Another protein targeted is TWIST that responsible for epithelial-mesenchymal transition and is related to angiogenesis, metastasis and drug resistance. So, using siRNA against TWIST protein conjugated with mesoporous silica nanoparticles (MSN-HAs) (siTWIST-MSN-HA) for delivery to epithelial OC (EOC) cells. HA help to specific target delivery to CD44 positive cells (A2780R cells). Moreover, due to the positive charge of PEI, the surface of MSN modified that, to improved attachment of the siRNA (negative charge) and HA to the amine groups in the PEI. By down-regulation of TWIST protein, OC cells become sensitive to drugs such as CIS. In vivo studies showed inhibition in tumor growth, and evaluating TWIST,

**183**

**Figure 5.**

*Nano Technology and Gas Plasma as Novel Therapeutic Strategies for Ovarian Cancer…*

Vimentin, N-Cadherin, and E-Cadherin tumor mRNA as EMT markers in mice that were treated by siTWIST-MSN-HA + CIS indicated great result in combination

Protein kinases are a considerable target for gene delivery for cancer treatment, so knockdown of p70 S6 kinase (p70S6K) is necessary for a decrease in migration, invasion and proliferation of OC stem cells (in vitro) and reduction in tumor growth and metastasis (in vivo). In this regard, p70S6K siRNA by G6 dendriplex NPs, that protect it from degradation, transfer to OC stem cells. Also, knockdown of p70S6K via this NP complex can inhibit the stemness and self-renewal properties

Kinesin spindle protein (KSP) is another gene, that by silencing, cell cycle arrest in mitotic phase and apoptosis happened in cancer cells. So, for transfection of KSP siRNA into the SKOV3 cells, PEGylated DC-Chol/DOPE lipoplexes were prepared. These NPs are caused to enhance accumulation in tumor tissue for suppression of tumor growth and decrease damage to kidneys and liver in SKOV3 tumor-bearing

Besides, growth and metastasis of tumor cells regulate by angiogenesis, so develop an NP with anti-angiogenesis property, plays an essential role in the treatment of OC. HA attached to chitosan NP enclose PLXDC1 siRNA for inhibition PLXDC1 as an angiogenesis gene. HUVEC and MOEC cells via expression CD44 have the potential to absorbed these nanocarriers and induced cytotoxicity and apoptosis. Administration HA-CH-NP/siRNA by A2780 tumor-bearing mice demonstrated antitumor characterization that causes to suppressing tumor growth

In another method for gene delivery, NPs were designed to transfer two siRNA to cancer cells to obtain the higher output. For instance, PLGA NPs loaded MDR1 and BCL2 siRNA were prepared. Silencing both genes simultaneously have an extraordinary effect on resistant OC cell sensitivity to PTX and CIS. In vitro experiments

*Targeting p70S6K with dendriplex nanoparticles inhibit stemness and metastatic properties of OC cells. This figure was obtained with permission from [51] under the terms of creative commons CC BY license.*

*DOI: http://dx.doi.org/10.5772/intechopen.96387*

of cancer stem cells (**Figure 5**) [51].

therapy [50].

mice [52].

and volume [53].

*Nano Technology and Gas Plasma as Novel Therapeutic Strategies for Ovarian Cancer… DOI: http://dx.doi.org/10.5772/intechopen.96387*

Vimentin, N-Cadherin, and E-Cadherin tumor mRNA as EMT markers in mice that were treated by siTWIST-MSN-HA + CIS indicated great result in combination therapy [50].

Protein kinases are a considerable target for gene delivery for cancer treatment, so knockdown of p70 S6 kinase (p70S6K) is necessary for a decrease in migration, invasion and proliferation of OC stem cells (in vitro) and reduction in tumor growth and metastasis (in vivo). In this regard, p70S6K siRNA by G6 dendriplex NPs, that protect it from degradation, transfer to OC stem cells. Also, knockdown of p70S6K via this NP complex can inhibit the stemness and self-renewal properties of cancer stem cells (**Figure 5**) [51].

Kinesin spindle protein (KSP) is another gene, that by silencing, cell cycle arrest in mitotic phase and apoptosis happened in cancer cells. So, for transfection of KSP siRNA into the SKOV3 cells, PEGylated DC-Chol/DOPE lipoplexes were prepared. These NPs are caused to enhance accumulation in tumor tissue for suppression of tumor growth and decrease damage to kidneys and liver in SKOV3 tumor-bearing mice [52].

Besides, growth and metastasis of tumor cells regulate by angiogenesis, so develop an NP with anti-angiogenesis property, plays an essential role in the treatment of OC. HA attached to chitosan NP enclose PLXDC1 siRNA for inhibition PLXDC1 as an angiogenesis gene. HUVEC and MOEC cells via expression CD44 have the potential to absorbed these nanocarriers and induced cytotoxicity and apoptosis. Administration HA-CH-NP/siRNA by A2780 tumor-bearing mice demonstrated antitumor characterization that causes to suppressing tumor growth and volume [53].

In another method for gene delivery, NPs were designed to transfer two siRNA to cancer cells to obtain the higher output. For instance, PLGA NPs loaded MDR1 and BCL2 siRNA were prepared. Silencing both genes simultaneously have an extraordinary effect on resistant OC cell sensitivity to PTX and CIS. In vitro experiments

### **Figure 5.**

*Ovarian Cancer - Updates in Tumour Biology and Therapeutics*

anti-apoptotic markers were evaluated [46].

improving delivery is also utilized in this technique.

**3.3 Gene delivery**

*3.3.1 NPs for siRNA delivery*

NP-drug conjugates (NDCs) are more effective than antibody-drug conjugates

Depolarization of mitochondria and augment the level of ROS that cause apoptosis and finally, cytotoxicity in tumor cells are other results of administration NPs individually or in combination with chemotherapy drugs. So, Gold NPs encapsulate theaflavin (tea-extracted polyphenols) (AuNP@TfQ ) as an apoptosis inducer in tumor cells. Anti-cancer activity of AuNP@TfQ enhanced by pristine theaflavin oxidation to its quinone derivative on the surface of gold NPs. The entrance of AuNP@TfQ into the PA-1 cells takes place through endocytosis. In this study caspase-3, Bax, Bad, BID, and BIM as pro-apoptotic markers and Bcl-2 and Bcl-was

But NPs individually can lead to cytotoxicity and apoptosis in tumor cells too. Gurunathan and colleagues proposed Ag NPs and ZnO NPs with a broad range of application in biomedical was used to treat OC individually and synergy with gemcitabine (GEM). Results represented a reduction in cell viability in a dose and time dependent manner and DNA double-strand break due to the overproduction

Herein, we focused on some gene delivery based examples used for OC treatment. NPs due to their properties are used as a vehicle for delivery of different nucleic acids such as siRNA, miRNA and shRNA. Increasing cytotoxicity by inducing apoptosis and suppressing tumor growth and volume are the common results obtain via silencing or downregulating oncogenes. Modifying NPs with ligands for

Using siRNA in oncotherapy because of low toxicity, high effectiveness and specificity is a terrific choice. To improve the efficiency and overcome the problem such as degradation by RNase and lack of the ability to penetrate the membrane cells, is better not to use naked siRNA. To facilitate the capability of siRNA and presence in proper concentration for silencing or downregulating of oncogenes, gene delivery systems play an essential role. Polymeric and lipid-based NPs are the most usable carriers for siRNA. Polymeric NPs especially PEI, PEG and chitosan with a positive charge easily bind to oligonucleotides with a negative charge through electrostatic interactions. While lipid NPs such as liposomes encapsulate siRNA in its aqueous core [30]. Polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol) modified FA (hyPEI-g-PCL-b-PEG-FA) is one of the examples polymeric NPs was

used for transfer siRNA to SKOV-3/LUC cells with a high level of FRα [49].

Another protein targeted is TWIST that responsible for epithelial-mesenchymal transition and is related to angiogenesis, metastasis and drug resistance. So, using siRNA against TWIST protein conjugated with mesoporous silica nanoparticles (MSN-HAs) (siTWIST-MSN-HA) for delivery to epithelial OC (EOC) cells. HA help to specific target delivery to CD44 positive cells (A2780R cells). Moreover, due to the positive charge of PEI, the surface of MSN modified that, to improved attachment of the siRNA (negative charge) and HA to the amine groups in the PEI. By down-regulation of TWIST protein, OC cells become sensitive to drugs such as CIS. In vivo studies showed inhibition in tumor growth, and evaluating TWIST,

of ROS and mitochondria dysfunction in both experiments [47, 48].

(ADCs) for loading monomethyl auristatin E (MMAE) in OC therapy. So, the results were very promising in the level of in vitro and in vivo, and inhibition of tumor growth and cytotoxicity in a patient-derived xenograft model of platinum-

resistant OC is twice as much in comparison with CIS administration [45].

**182**

*Targeting p70S6K with dendriplex nanoparticles inhibit stemness and metastatic properties of OC cells. This figure was obtained with permission from [51] under the terms of creative commons CC BY license.*

implement on the PTX-resistant and CIS-resistant, SKOV3-TR and A2780-CP20 cells respectively. The observations indicated an increment in cellular uptake that induces cell death by apoptosis and necrosis [54].

## *3.3.2 NPs for shRNA delivery*

shRNA is a stem-loop RNA that in comparison with siRNA cause prolong gene silencing and highly effective. In the following, 2 examples of this procedure have been brought. PEG NPs with a peptide of FSH β 33-53 for specific target delivery encapsulate shRNA for silencing growth-regulated oncogene α (gro-α) (FSH33- G-NP). Internalization in FSHR positive cells like HEY cells is more. FSH33-G-NP decrement cell proliferation, invasion and migration and also in vivo experiments showed antitumor activity [55]. Also, overexpression of pin1 is related to cancer malignancy by regulating oncogenes and tumor suppressor genes, so silencing of pin1 can inhibit the tumor growth in a syngeneic mouse model and induce apoptosis in OC cells. Proteasome-dependent degradation of Pin1 happened via liposomebased NPs that were modified by cyclodextrins for shRNA delivery (**Figure 6**) [56].
