As an anthracycline anticancer agent, **epirubicin** has a wide range of antitumor activity and is used to treat various carcinomas. However, this therapy may cause some serious side effects such as allergic reactions, cardiotoxicity and blood problems. Therefore, nanoparticles being used as a carrier of epirubicin are hoped to sustain its release, prolong its circulation time, enhance its therapeutic index and decrease its toxic effects. Cholesterolmodified nanoparticles have been prepared by self-assembling method by Wang and coworkers (Wang et al., 2007). They synthesized cholesterol-modified chitosan conjugate with succinyl linkages, investigated its self-aggregation behaviour, and prepared self-aggregated nanoparticles by sonication in aqueous media. Nanoparticles loading efficiency varied from 25 to 71% depending on the weight ratio of epirubicin and the nanoparticles and exhibited the release profile relating to the pH of the release media. When the pH of the release media increased, an evident decrease of drug release rate was observed owing to the solubility of epirubicin is greatly influenced by the pH of the aqueous solution, and self-aggregated nanoparticles were also pH sensitive because of the presence of many amino groups in their molecules. Thus, the drug release from self-aggregated nanoparticles was very slow in PBS (pH 7.4) and the total release amount was about 25% in 48 h, which suggested self-

aggregated nanoparticles had a potential as a sustained-release carrier of epirubicin.

**systems**

**4.3 Nucleic acids and genetic material included in polysaccharide-based delivery**

**Small interfering RNAs** (siRNAs) have proven to be versatile agents for controlling gene expression in mammalian cells. They have been employed as a novel tool since they can block the expression of genes, such as those expressed in infectious diseases and cancers.

thus an improvement in therapeutic efficacy (Zhang et al., 2007).

loaded nanoparticles were prepared by Sarmah and co-workers based on guar gum, which is commonly used for colon specific drug delivery in the pharmaceutical industry. Nanoparticles were obtained by o/w emulsification and in situ polymer cross-linking, using dichloromethane as the best solvent of the drug and glutaraldehyde as cross-linker agent during the process. An efficiency loading of 15% was obtained when dichloromethane was used as selected solvent (Sarmah et al., 2009). Recently, nanoparticles based on thiolated alginate (ALG-CYS) and disulfide bond reduced albumin (BSA-SH) have been synthesized by coacervation method and stabilized by disulfide bond formation between both polymers. *In vitro* studies revealed that total release of the drug was not achieved in any case; only the 23–61% of the drug was released. Maximum release took place between 7 and 75 h. According to the results, it was concluded that the presence of alginate in the nanoparticle composition allowed the modulation of the amount of released TMX (Martínez et al., 2011).

**Mitoxantrone** is often used to treat breast cancer clinically, but the prolonged treatment with this drug results in some side-effects, such as heart toxicity and myelosupression, which are often a problem. Mitoxantrone is positively charged and it can be absorbed by negatively charged polysaccharides, such as chitosan. Nanospheres can be obtained by ion gelation method using sodium TPP as gelation agent, and obtaining an encapsulation efficacy of 98%. Tests for *in vitro* release in physiological saline or physiological saline containing 0.5% (w/v) ascorbic acid by a dialysis bag showed sustained release and little burst effect (Lu et al., 2006).

**Methotrexate (MTX)** is a folate antimetabolite and has been used in the treatment of various malignancies, including childhood acute lymphocytic leukemia, osteosarcoma, non-Hodgkin's lymphoma, Hodgkin's disease, head and neck cancer, lung cancer, and breast cancer. However, it may cause some adverse effects such as bone marrow suppression, acute and chronic hepatotoxicity, interstitial pneumonitis and chronic interstitial obstructive pulmonary disease. Therefore, in order to reduce its toxic and side effects and to improve specificity and selectivity, it is a good candidate to be included in drug delivery systems. Yang and co-workers have designed methoxy poly(ethylene glycol)-grafted-chitosan (mPEG-g-CS) self-aggregated nanoparticles being used as a carrier of MTX . Depending on the formulation, loading efficiency varied from 21 to 95%. *In vitro* release studies showed that the MTX appeared to be released in a biphasic way, which characterized by an initial release or rapid release period followed by a step of slower release. A fast release was observed in 4 h, in which 40% of the drug was released from nanoparticles. After this initial effect, MTX was released in a continuous way for up to 48 h, reaching percentage of cumulative release close to 60%. Therefore, the self-aggregated nanoparticles delayed the drug release in the release process when these results were compared with the release of the free drug (Yang et al., 2008).

**Doxorubicin (DOX)** is a member of the anthracycline ring antibiotics, with a broad spectrum of antitumor activity, including a variety of human and animal solid tumors. Despite advances of this antitumor agent, it is hydrophobic and possesses inevitable, serious side effects such as nonspecific toxicity that limit the dose and use of the drug. Therefore, a lot of studies have been carried out in order to entrap this drug into different drug carriers. DOX has been successfully entrapped into oleoyl-chitosan nanoparticles prepared by o/w emulsification method. Nanoparticles had a high encapsulation efficiency of 53% in loading doxorubicin, being drug incorporation into nanoparticles generally limited by the large

loaded nanoparticles were prepared by Sarmah and co-workers based on guar gum, which is commonly used for colon specific drug delivery in the pharmaceutical industry. Nanoparticles were obtained by o/w emulsification and in situ polymer cross-linking, using dichloromethane as the best solvent of the drug and glutaraldehyde as cross-linker agent during the process. An efficiency loading of 15% was obtained when dichloromethane was used as selected solvent (Sarmah et al., 2009). Recently, nanoparticles based on thiolated alginate (ALG-CYS) and disulfide bond reduced albumin (BSA-SH) have been synthesized by coacervation method and stabilized by disulfide bond formation between both polymers. *In vitro* studies revealed that total release of the drug was not achieved in any case; only the 23–61% of the drug was released. Maximum release took place between 7 and 75 h. According to the results, it was concluded that the presence of alginate in the nanoparticle composition allowed the modulation of the amount of released TMX (Martínez et al., 2011). **Mitoxantrone** is often used to treat breast cancer clinically, but the prolonged treatment with this drug results in some side-effects, such as heart toxicity and myelosupression, which are often a problem. Mitoxantrone is positively charged and it can be absorbed by negatively charged polysaccharides, such as chitosan. Nanospheres can be obtained by ion gelation method using sodium TPP as gelation agent, and obtaining an encapsulation efficacy of 98%. Tests for *in vitro* release in physiological saline or physiological saline containing 0.5% (w/v) ascorbic acid by a dialysis bag showed sustained release and little

**Methotrexate (MTX)** is a folate antimetabolite and has been used in the treatment of various malignancies, including childhood acute lymphocytic leukemia, osteosarcoma, non-Hodgkin's lymphoma, Hodgkin's disease, head and neck cancer, lung cancer, and breast cancer. However, it may cause some adverse effects such as bone marrow suppression, acute and chronic hepatotoxicity, interstitial pneumonitis and chronic interstitial obstructive pulmonary disease. Therefore, in order to reduce its toxic and side effects and to improve specificity and selectivity, it is a good candidate to be included in drug delivery systems. Yang and co-workers have designed methoxy poly(ethylene glycol)-grafted-chitosan (mPEG-g-CS) self-aggregated nanoparticles being used as a carrier of MTX . Depending on the formulation, loading efficiency varied from 21 to 95%. *In vitro* release studies showed that the MTX appeared to be released in a biphasic way, which characterized by an initial release or rapid release period followed by a step of slower release. A fast release was observed in 4 h, in which 40% of the drug was released from nanoparticles. After this initial effect, MTX was released in a continuous way for up to 48 h, reaching percentage of cumulative release close to 60%. Therefore, the self-aggregated nanoparticles delayed the drug release in the release process when these results were compared with the release of the

**Doxorubicin (DOX)** is a member of the anthracycline ring antibiotics, with a broad spectrum of antitumor activity, including a variety of human and animal solid tumors. Despite advances of this antitumor agent, it is hydrophobic and possesses inevitable, serious side effects such as nonspecific toxicity that limit the dose and use of the drug. Therefore, a lot of studies have been carried out in order to entrap this drug into different drug carriers. DOX has been successfully entrapped into oleoyl-chitosan nanoparticles prepared by o/w emulsification method. Nanoparticles had a high encapsulation efficiency of 53% in loading doxorubicin, being drug incorporation into nanoparticles generally limited by the large

burst effect (Lu et al., 2006).

free drug (Yang et al., 2008).

surface area of the latter, as well as by the solubility of the drug in water. The drug was completely released from nanoparticles in the buffer medium of Na2HPO4– citric acid (pH 3.8), whereas in PBS (pH 7.4) 65% of doxorubicin was released after 6 hours, followed by a sustained release until 72 hours. Approximately 72% of DOX was released for 3 days, showing the potential of the nanoparticles as a sustained drug delivery system, suggesting that the nanoparticles might act as a barrier against the release of entrapped DOX. This result indicated that the nanoparticles contributed to an extended circulation of DOX and thus an improvement in therapeutic efficacy (Zhang et al., 2007).

**Paclitaxel** is an anticancer drug which is used in different types of malignancies, such as ovarian and breast cancer. Despite its multiple applications, it has important problems of solubility in water and, consequently, its administration is carried out including an oily component (Cremophor) and dehydrated alcohol within its formulation. Thus, relevant side effects such as hypersensibility, hypotension or breast pain are frequently observed after its intravenous administration. In the last years, a lot of studies have included this drug as good candidate to be included in drug delivery systems to overcome its administration problems. Hu and coworkers have obtained paclitaxel-loaded nanoparticles by self-crosslinking stearic-grafted chitosan and using glutaraldehyde as cross-linker in order to stabilize the systems. These formulations showed high encapsulation efficiency which ranged from 95% to 99%. When the surfaces of micelles were cross-linked by glutaraldehyde, the burst release of the micelles at earlier stage was highly improved and the drug release time was prolonged. By controlling the amino substitution of stearic-grafted chitosan and the crosslink degree, the prolonged and controlled release could be achieved (Hu et al., 2006).

As an anthracycline anticancer agent, **epirubicin** has a wide range of antitumor activity and is used to treat various carcinomas. However, this therapy may cause some serious side effects such as allergic reactions, cardiotoxicity and blood problems. Therefore, nanoparticles being used as a carrier of epirubicin are hoped to sustain its release, prolong its circulation time, enhance its therapeutic index and decrease its toxic effects. Cholesterolmodified nanoparticles have been prepared by self-assembling method by Wang and coworkers (Wang et al., 2007). They synthesized cholesterol-modified chitosan conjugate with succinyl linkages, investigated its self-aggregation behaviour, and prepared self-aggregated nanoparticles by sonication in aqueous media. Nanoparticles loading efficiency varied from 25 to 71% depending on the weight ratio of epirubicin and the nanoparticles and exhibited the release profile relating to the pH of the release media. When the pH of the release media increased, an evident decrease of drug release rate was observed owing to the solubility of epirubicin is greatly influenced by the pH of the aqueous solution, and self-aggregated nanoparticles were also pH sensitive because of the presence of many amino groups in their molecules. Thus, the drug release from self-aggregated nanoparticles was very slow in PBS (pH 7.4) and the total release amount was about 25% in 48 h, which suggested selfaggregated nanoparticles had a potential as a sustained-release carrier of epirubicin.

#### **4.3 Nucleic acids and genetic material included in polysaccharide-based delivery systems**

**Small interfering RNAs** (siRNAs) have proven to be versatile agents for controlling gene expression in mammalian cells. They have been employed as a novel tool since they can block the expression of genes, such as those expressed in infectious diseases and cancers.

Polysaccharide-Based Nanoparticles for Controlled Release Formulations 211

gelation method, microparticles made of alginate cross-linked with calcium ions and poly-Llysine have been reported to effectively act as transfecting agents. Encapsulation efficacies of 32-95% depending on the composition of the formulation have been obtained and the *in vitro* release behaviour depended mostly on the medium composition. The importance of competitive anions and ionic strength on the mechanism of dissociation of the oligonucleotide from the polymeric matrix was observed. Thus, the presence of phosphate anions preferably displaced alginate from the structure, resulting in the release of complexed oligonucleotide. Rat *in vivo* studies showed promising oligonucleotide bioavailability for microparticles after intrajejunal administration in the presence of a mixture of permeation enhancers to achieve a successful intestinal application (Gonzalez

Natural biopolymers are also widely used in the field of **gene delivery**. In fact, alginate nanoparticles have been prepared using w/o microemulsion as a template followed by calcium cross-linking of guluronic acid units of alginate polymer. Ca-alginate nanoparticles were loaded with GFP-encoding plasmids in order to study their potency as carriers for gene delivery. The degree of endocytosis by NIH 3T3 cells and ensuing transfection rate were investigated. Results showed that Ca-alginate nanoparticles were very efficient gene

**Furosemide** is a loop diuretic used in the treatment of congestive heart failure and edema, and it has been recently incorporated into chitosan nanoparticles (Zhi et al., 2005). Since the chitosan molecule has strong interaction with the organic compounds, it can be applied to adsorb diuretics from the water samples. Zhi and coworkers prepared chitosan nanoparticles by a nanoemulsion system, in which the chitosan nanoparticles were prepared by adding NaOH solution or glutaraldehyde as the solidification solution. Size was smaller in case of using NaOH as cross-linker agent (30 - 150 nm). The adsorptive efficiency of furosemide on the nanoparticle was 51.9%, and the presence of less cross-linking agent could be in favour of the adsorption ability. The furosemide adsorption capacity on the chitosan nanoparticles was affected not only by the –NH2 content, but also by the molecular

Ophthalmic drug delivery, probably more than any other route of administration, may benefit from the characteristics of nanotechnology-based drug delivery systems, mainly because of their capacity to protect the encapsulated molecule while facilitating its transport to the different compartments of the eye (Raju & Goldberg, 2008). For this purpose, the common use of chitosan is justified by its mucoadhesive and penetration enhancing properties, as well as by its good biocompatibility with the ocular structures. **Cyclosporine A (CyA)** is an immunosuppressant drug widely used in post-allogeneic organ transplant to reduce the activity of the immune system, and therefore the risk of organ rejection. The entrapment of the hydrophobic polypeptide CyA was achieved by ionic gelation technique using TPP and a previous dissolution of the peptide in an acetonitrile: water mixture, and a further nanoprecipitation into the nanoparticles in the form of small nanocrystals (de la Fuente et al., 2010). Entrapment efficiencies were reported to be as high as 73.4%. The *in vivo* evaluation of this new prototype, in rabbits, evidenced the capacity of these nanoparticles providing a selective and prolonged delivery of CyA to the cornea and conjunctiva. More

**4.4 Other drugs included in polysaccharide-based delivery systems**

Ferreiro et al., 2002).

carriers (You & Peng, 2004).

weight of chitosan.

However, siRNA suffers particular problems including poor cellular uptake, rapid degradation as well as limited blood stability. Therefore, effective systems which can protect and transport siRNA to the cytoplasm of the targeted cells are needed to exploit the promising potential applications offered by successful delivery of siRNA. Chitosan has been used to prepare nanoparticles based on modified ionic gelation with TPP as cross-linker agent, and they have been applied as possible vector that should be able to be taken up by the cells and escape the endosomal vesicle to avoid lysosomal degradation. *In vitro* study revealed the transfection efficiency of siRNA depends on the method of siRNA association to the chitosan, and entrapping siRNA using ionic gelation has shown to yield a better biological effect than simple complexation or siRNA adsorption onto the chitosan nanoparticles (Katas & Alpar, 2006).

Ionotropic complexation (PEC) between chitosan and different polyanions has been used as an useful method of preparation of nanoparticles in order to include nucleic acids, since chitosan-**DNA** nanoparticles demonstrated low transfection efficiencies and the incorporation of secondary polymers improved the characteristics of these systems (Kaul & Amiji, 2002). Among the chitosan–polyanion complexes investigated, the combination of chitosan and alginate is considered to be among the most interesting for delivery systems. The method used to prepare the nanoparticles is a two-step method where the first step is the formation of a calcium–alginate pre-gel. Various concentrations of chitosan solutions were then added with continuous stirring. High loading efficacy was obtained (26-60%), while maximum mass loading was 60 *μ*g DNA/mg nanoparticles and 6% and 3.5% of the adsorbed DNA was released. Therefore, the high encapsulation of DNA from alginate– chitosan nanoparticles is encouraging for application in the field of gene therapy (Douglas & Tabrizian, 2005). The complexation between chitosan and carboxymethylcellulose (CMC) is another PEC combination employed in the inclusion of nucleic acid into delivery systems. Cui and Mumper coated the surface of this pre-formed nanoparticles with plasmid-DNA, obtaining a final plasmid DNA concentration of up to 400 mg/ml. Chitosan/CMC based nanoparticles containing plasmid DNA were applied topically to the skin of shaved mice and resulted in detectable and quantifiable levels of luciferase expression in skin after 24 h (Cui & Mumper, 2001). Nucleic acids can be also combined with chitosan in order to obtain nanospheres, being part of the structure of the system at the same time they are the encapsulant of the nanospheres. Chitosan showed a very high protection efficiency for siRNA. Polyplexes prepared with this polysaccharide are able to protect a siRNA for 7 h and an efficient transfection of the cells may be obtained after the intranasal administration of siRNA/chitosan polyplexes (de Martimprey et al., 2009).

**Antisense oligonucleotides** are therapeutic agents known to selectively modulate gene expression. The development of non-parenteral dosage forms for these compounds is desirable. However, the high molecular weight, the hydrophilicity and multiple negative charges result in a poor absorption of antisense oligonucleotides. Moreover, the oral administration faces additional problems such as degradation in the acidic gastric environment, enzymatic metabolism in the lumen and at the gastrointestinal epithelium and first-pass hepatic clearance. To achieve a successful non-parenteral delivery of antisense therapeutics, it is necessary to solve the specific problems of the oral administration route, together with general concerns of correct time-space targeting, improved cellular uptake and nuclear localization to exert gene transfection (Akhtar et al., 2000). Using a ionotropic

However, siRNA suffers particular problems including poor cellular uptake, rapid degradation as well as limited blood stability. Therefore, effective systems which can protect and transport siRNA to the cytoplasm of the targeted cells are needed to exploit the promising potential applications offered by successful delivery of siRNA. Chitosan has been used to prepare nanoparticles based on modified ionic gelation with TPP as cross-linker agent, and they have been applied as possible vector that should be able to be taken up by the cells and escape the endosomal vesicle to avoid lysosomal degradation. *In vitro* study revealed the transfection efficiency of siRNA depends on the method of siRNA association to the chitosan, and entrapping siRNA using ionic gelation has shown to yield a better biological effect than simple complexation or siRNA adsorption onto the chitosan

Ionotropic complexation (PEC) between chitosan and different polyanions has been used as an useful method of preparation of nanoparticles in order to include nucleic acids, since chitosan-**DNA** nanoparticles demonstrated low transfection efficiencies and the incorporation of secondary polymers improved the characteristics of these systems (Kaul & Amiji, 2002). Among the chitosan–polyanion complexes investigated, the combination of chitosan and alginate is considered to be among the most interesting for delivery systems. The method used to prepare the nanoparticles is a two-step method where the first step is the formation of a calcium–alginate pre-gel. Various concentrations of chitosan solutions were then added with continuous stirring. High loading efficacy was obtained (26-60%), while maximum mass loading was 60 *μ*g DNA/mg nanoparticles and 6% and 3.5% of the adsorbed DNA was released. Therefore, the high encapsulation of DNA from alginate– chitosan nanoparticles is encouraging for application in the field of gene therapy (Douglas & Tabrizian, 2005). The complexation between chitosan and carboxymethylcellulose (CMC) is another PEC combination employed in the inclusion of nucleic acid into delivery systems. Cui and Mumper coated the surface of this pre-formed nanoparticles with plasmid-DNA, obtaining a final plasmid DNA concentration of up to 400 mg/ml. Chitosan/CMC based nanoparticles containing plasmid DNA were applied topically to the skin of shaved mice and resulted in detectable and quantifiable levels of luciferase expression in skin after 24 h (Cui & Mumper, 2001). Nucleic acids can be also combined with chitosan in order to obtain nanospheres, being part of the structure of the system at the same time they are the encapsulant of the nanospheres. Chitosan showed a very high protection efficiency for siRNA. Polyplexes prepared with this polysaccharide are able to protect a siRNA for 7 h and an efficient transfection of the cells may be obtained after the intranasal administration of

**Antisense oligonucleotides** are therapeutic agents known to selectively modulate gene expression. The development of non-parenteral dosage forms for these compounds is desirable. However, the high molecular weight, the hydrophilicity and multiple negative charges result in a poor absorption of antisense oligonucleotides. Moreover, the oral administration faces additional problems such as degradation in the acidic gastric environment, enzymatic metabolism in the lumen and at the gastrointestinal epithelium and first-pass hepatic clearance. To achieve a successful non-parenteral delivery of antisense therapeutics, it is necessary to solve the specific problems of the oral administration route, together with general concerns of correct time-space targeting, improved cellular uptake and nuclear localization to exert gene transfection (Akhtar et al., 2000). Using a ionotropic

nanoparticles (Katas & Alpar, 2006).

siRNA/chitosan polyplexes (de Martimprey et al., 2009).

gelation method, microparticles made of alginate cross-linked with calcium ions and poly-Llysine have been reported to effectively act as transfecting agents. Encapsulation efficacies of 32-95% depending on the composition of the formulation have been obtained and the *in vitro* release behaviour depended mostly on the medium composition. The importance of competitive anions and ionic strength on the mechanism of dissociation of the oligonucleotide from the polymeric matrix was observed. Thus, the presence of phosphate anions preferably displaced alginate from the structure, resulting in the release of complexed oligonucleotide. Rat *in vivo* studies showed promising oligonucleotide bioavailability for microparticles after intrajejunal administration in the presence of a mixture of permeation enhancers to achieve a successful intestinal application (Gonzalez Ferreiro et al., 2002).

Natural biopolymers are also widely used in the field of **gene delivery**. In fact, alginate nanoparticles have been prepared using w/o microemulsion as a template followed by calcium cross-linking of guluronic acid units of alginate polymer. Ca-alginate nanoparticles were loaded with GFP-encoding plasmids in order to study their potency as carriers for gene delivery. The degree of endocytosis by NIH 3T3 cells and ensuing transfection rate were investigated. Results showed that Ca-alginate nanoparticles were very efficient gene carriers (You & Peng, 2004).

#### **4.4 Other drugs included in polysaccharide-based delivery systems**

**Furosemide** is a loop diuretic used in the treatment of congestive heart failure and edema, and it has been recently incorporated into chitosan nanoparticles (Zhi et al., 2005). Since the chitosan molecule has strong interaction with the organic compounds, it can be applied to adsorb diuretics from the water samples. Zhi and coworkers prepared chitosan nanoparticles by a nanoemulsion system, in which the chitosan nanoparticles were prepared by adding NaOH solution or glutaraldehyde as the solidification solution. Size was smaller in case of using NaOH as cross-linker agent (30 - 150 nm). The adsorptive efficiency of furosemide on the nanoparticle was 51.9%, and the presence of less cross-linking agent could be in favour of the adsorption ability. The furosemide adsorption capacity on the chitosan nanoparticles was affected not only by the –NH2 content, but also by the molecular weight of chitosan.

Ophthalmic drug delivery, probably more than any other route of administration, may benefit from the characteristics of nanotechnology-based drug delivery systems, mainly because of their capacity to protect the encapsulated molecule while facilitating its transport to the different compartments of the eye (Raju & Goldberg, 2008). For this purpose, the common use of chitosan is justified by its mucoadhesive and penetration enhancing properties, as well as by its good biocompatibility with the ocular structures. **Cyclosporine A (CyA)** is an immunosuppressant drug widely used in post-allogeneic organ transplant to reduce the activity of the immune system, and therefore the risk of organ rejection. The entrapment of the hydrophobic polypeptide CyA was achieved by ionic gelation technique using TPP and a previous dissolution of the peptide in an acetonitrile: water mixture, and a further nanoprecipitation into the nanoparticles in the form of small nanocrystals (de la Fuente et al., 2010). Entrapment efficiencies were reported to be as high as 73.4%. The *in vivo* evaluation of this new prototype, in rabbits, evidenced the capacity of these nanoparticles providing a selective and prolonged delivery of CyA to the cornea and conjunctiva. More

Polysaccharide-Based Nanoparticles for Controlled Release Formulations 213

AmB exhibited only moderate interaction with the weakly cross-linked polymers of the nanoparticles. A reduction of nephrotoxicity was observed in an *in vivo* renal toxicity study.

As pointed out throughout this review, polysaccharides show variability and versatility, due to their complex structure, which is difficult to be reproduced with synthetic polymers. Thus, native polysaccharides and their derivatives are emerging in the last years as one of the most used biomaterials in the field of nanomedicine, especially being chosen by a lot of researchers as carriers to be used in the preparation of nanoparticulate drug delivery

A wide variety of preparation methods of nanoparticles has been developed, and three aspects have marked the evolution of these methods: need for lees toxic agents, simplification of the procedures and optimization to improve yield and entrapment efficiency. Now it is possible to choose the best method of preparation and the best suitable polymer to achieve an efficient encapsulation of the drug, taking into account the drug

As reviewed above, so many nanoparticle drug delivery systems have been prepared using various polysaccharides as carriers combined with different groups of drugs, and they have been investigated in terms of physicochemical features, drug -loading efficiency, *in vitro* toxicity and comparative *in vivo* test. Deeper studies, such as evaluation of interaction between cells, tissues and organs, as well as how the administration of these systems can affect to the metabolism, need to be carried out. In fact, more and more nanoparticle systems are emerging nowadays, and these necessary studies will be focused on in the near future,

The financial support of the Ministerio de Ciencia e Innovación of Spain FIS(PS09/01513) and MAT2010-21509-C03-03, and the FPI grant from UCM to A. Martínez are gratefully

Ahmad, Z.; Pandey, R.; Sharma, S. & Khuller, G. K. (2006). Pharmacokinetic and

Akbuga, J. & Durmaz, G. (1994). Preparation and evaluation of crosslinked chitosan

Akhtar, S.; Hughes, M. D.; Khan, A.; Bibby, M.; Hussain, M.; Nawaz, Q.; Double, J. &

Akiyoshi, K.; Kobayashi, S.; Shichibe, S.; Mix, D.; Baudys, M.; Kim, S. W. & Sunamoto, J.

pp. (3-21), 0169-409X (Print) 0169-409X (Linking).

pharmacodynamic behaviour of antitubercular drugs encapsulated in alginate nanoparticles at two doses. *Int J Antimicrob Agents*, 27, 5, pp. (409-416), 0924-8579

microspheres containing furosemide. *International Journal of Pharmaceutics*, 11, pp.

Sayyed, P. (2000). The delivery of antisense therapeutics. *Adv Drug Deliv Rev*, 44, 1,

(1998). Self-assembled hydrogel nanoparticle of cholesterol-bearing pullulan as a

completing the evaluation of these hopeful polysaccharide-based systems.

**5. Conclusion**

features in this selection.

**6. Acknowledgements**

(217-222).

(Print) 0924-8579 (Linking).

acknowledged.

**7. References**

systems.

importantly, it was observed that CyA-loaded chitosan nanoparticles provided therapeutic levels of CyA in the conjunctiva and the cornea for up to 24 and 48 h postadministration, respectively, while reducing the access of CyA to the blood circulation. This positive behavior of chitosan nanoparticles was attributed to their improved interaction with the corneal and conjunctival and the prolonged delivery of the CyA molecules associated to them (de la Fuente et al., 2010).

**Retinol** and its derivatives are extensively used in the pharmaceutical and cosmetic area. Especially, retinoids are recognized as being important for modern therapy of dermatological treatment of wrinkled skin. The stability and solubility problems of these compouns make them ideal candidates to be included into carriers. Nanoparticles are reported to be useful formulation to solve the poor aqueous solubility of retinoids and are able to use it by intravenous injection. Chitosan has been used by Kim and coworkers in order to obtain retinol-encapsulated chitosan nanoparticles for application of cosmetic and pharmaceutical applications (Kim et al., 2006). Retinol-encapsulated nanoparticles were completely reconstituted into aqueous solution as same as original aqueous solution.

The requirement of multidrug administration daily or several times a week for at least 6 months is the main cause of the failure of antitubercular chemotherapy. The dose-dependent side effects with high incidence and the need of daily-dosing results in discontinuation of medication, relapse of symptoms and an alarming increase in the prevalence of multidrugresistant strains (Pandey & Khuller, 2005). Consequently, reductions in dose and dosing frequency are the major goals of tuberculosis research and the application of drug delivery systems constitutes an important therapeutic strategy. **Isoniazid, pyrazinamide and rifampicin**, which are important antitubercular drugs, were encapsulated into alginate nanoparticles prepared by cation-induced gelification, reaching drug encapsulation efficiencies of 70–90% for isoniazid and pyrazinamide and 80–90% for rifampicin (Ahmad et al., 2006). When alginate nanoparticles were administered through nebulisation, the drugs were detected in plasma from 3 h onwards. Encapsulated drugs were observed up to 14, 10 and 14 days, respectively, in contrast with free drugs that were cleared from the circulation within 12–24 h. The levels of drugs in various organs remained above the minimum inhibitory concentration at both doses for equal periods, demonstrating their equiefficiency. Alginate nanoparticles hold great potential in reducing dosing frequency of antitubercular drugs.

**Amphotericin B** (AmB) is a polyene macrolide antifungal agent and the drug of choice for systemic fungal infection. Unfortunately, it is poorly absorbed from the gastrointestinal tract due to its low aqueous solubility and it must be given parenterally to treat systemic fungal infections. Currently, two types of drug formulations for AmB are available. The first one is a micellar solution of AmB and the second one are lipid-based nanoparticulate formulations. Despite these formulations reduce nephrotoxicity of the treatment, they are still quite expensive. Thus, much effort has been spent to develop cheaper delivery systems with reduced amphotericin B toxicity. Polysaccharide based nanoparticle delivery systems are one approach that has been investigated by Tiyaboonchai and coworkers (Tiyaboonchai & Limpeanchob, 2007). They prepared AmB-chitosan-dextrane sulfate nanoparticles by polyelectrolyte complexation at room temperature, showing an an association efficiency of 50–65%. They observed a fast release characteristic of AmB independent of the processing conditions, with most of AmB released from particles within 5 min. This suggested that AmB exhibited only moderate interaction with the weakly cross-linked polymers of the nanoparticles. A reduction of nephrotoxicity was observed in an *in vivo* renal toxicity study.

### **5. Conclusion**

212 The Delivery of Nanoparticles

importantly, it was observed that CyA-loaded chitosan nanoparticles provided therapeutic levels of CyA in the conjunctiva and the cornea for up to 24 and 48 h postadministration, respectively, while reducing the access of CyA to the blood circulation. This positive behavior of chitosan nanoparticles was attributed to their improved interaction with the corneal and conjunctival and the prolonged delivery of the CyA molecules associated to

**Retinol** and its derivatives are extensively used in the pharmaceutical and cosmetic area. Especially, retinoids are recognized as being important for modern therapy of dermatological treatment of wrinkled skin. The stability and solubility problems of these compouns make them ideal candidates to be included into carriers. Nanoparticles are reported to be useful formulation to solve the poor aqueous solubility of retinoids and are able to use it by intravenous injection. Chitosan has been used by Kim and coworkers in order to obtain retinol-encapsulated chitosan nanoparticles for application of cosmetic and pharmaceutical applications (Kim et al., 2006). Retinol-encapsulated nanoparticles were

completely reconstituted into aqueous solution as same as original aqueous solution.

The requirement of multidrug administration daily or several times a week for at least 6 months is the main cause of the failure of antitubercular chemotherapy. The dose-dependent side effects with high incidence and the need of daily-dosing results in discontinuation of medication, relapse of symptoms and an alarming increase in the prevalence of multidrugresistant strains (Pandey & Khuller, 2005). Consequently, reductions in dose and dosing frequency are the major goals of tuberculosis research and the application of drug delivery systems constitutes an important therapeutic strategy. **Isoniazid, pyrazinamide and rifampicin**, which are important antitubercular drugs, were encapsulated into alginate nanoparticles prepared by cation-induced gelification, reaching drug encapsulation efficiencies of 70–90% for isoniazid and pyrazinamide and 80–90% for rifampicin (Ahmad et al., 2006). When alginate nanoparticles were administered through nebulisation, the drugs were detected in plasma from 3 h onwards. Encapsulated drugs were observed up to 14, 10 and 14 days, respectively, in contrast with free drugs that were cleared from the circulation within 12–24 h. The levels of drugs in various organs remained above the minimum inhibitory concentration at both doses for equal periods, demonstrating their equiefficiency. Alginate nanoparticles hold great potential in reducing dosing frequency of antitubercular

**Amphotericin B** (AmB) is a polyene macrolide antifungal agent and the drug of choice for systemic fungal infection. Unfortunately, it is poorly absorbed from the gastrointestinal tract due to its low aqueous solubility and it must be given parenterally to treat systemic fungal infections. Currently, two types of drug formulations for AmB are available. The first one is a micellar solution of AmB and the second one are lipid-based nanoparticulate formulations. Despite these formulations reduce nephrotoxicity of the treatment, they are still quite expensive. Thus, much effort has been spent to develop cheaper delivery systems with reduced amphotericin B toxicity. Polysaccharide based nanoparticle delivery systems are one approach that has been investigated by Tiyaboonchai and coworkers (Tiyaboonchai & Limpeanchob, 2007). They prepared AmB-chitosan-dextrane sulfate nanoparticles by polyelectrolyte complexation at room temperature, showing an an association efficiency of 50–65%. They observed a fast release characteristic of AmB independent of the processing conditions, with most of AmB released from particles within 5 min. This suggested that

them (de la Fuente et al., 2010).

drugs.

As pointed out throughout this review, polysaccharides show variability and versatility, due to their complex structure, which is difficult to be reproduced with synthetic polymers. Thus, native polysaccharides and their derivatives are emerging in the last years as one of the most used biomaterials in the field of nanomedicine, especially being chosen by a lot of researchers as carriers to be used in the preparation of nanoparticulate drug delivery systems.

A wide variety of preparation methods of nanoparticles has been developed, and three aspects have marked the evolution of these methods: need for lees toxic agents, simplification of the procedures and optimization to improve yield and entrapment efficiency. Now it is possible to choose the best method of preparation and the best suitable polymer to achieve an efficient encapsulation of the drug, taking into account the drug features in this selection.

As reviewed above, so many nanoparticle drug delivery systems have been prepared using various polysaccharides as carriers combined with different groups of drugs, and they have been investigated in terms of physicochemical features, drug -loading efficiency, *in vitro* toxicity and comparative *in vivo* test. Deeper studies, such as evaluation of interaction between cells, tissues and organs, as well as how the administration of these systems can affect to the metabolism, need to be carried out. In fact, more and more nanoparticle systems are emerging nowadays, and these necessary studies will be focused on in the near future, completing the evaluation of these hopeful polysaccharide-based systems.

### **6. Acknowledgements**

The financial support of the Ministerio de Ciencia e Innovación of Spain FIS(PS09/01513) and MAT2010-21509-C03-03, and the FPI grant from UCM to A. Martínez are gratefully acknowledged.

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**10**

*USA*

**Electrospray Production of Nanoparticles**

*2William G. Lowrie Department of Chemical and Biomolecular Engineering,*

*3Department of Chemistry, The Ohio State University, Columbus, Ohio*

Yun Wu1, Anthony Duong1,2, L. James Lee1,2 and Barbara E. Wyslouzil1,2,3

Nanomedicine – the application of nanotechnology to medicine – shows great potential to positively impact healthcare. Nanoparticles, including solid lipid nanoparticles, lipoplexes and polyplexes, can act as carriers to deliver the drugs or nucleic acid-based therapeutics

Many techniques have been developed to produce nanoparticles. Among them, electrospray has attracted recent research interest because it is an elegant and versatile way to make a broad array of nanoparticles. Electrospray is a technique that uses an electric field to disperse or break up a liquid. Compared with current technologies, such as bulk mixing, high pressure homogenization and double emulsion techniques, electrospray has three potential advantages. First, electrospray can generate monodisperse droplets whose size can vary from tens of nanometer to hundreds of micrometers, depending on the processing parameters. Secondly, it is a very gentle method. The free charge, induced by the electric field, only concentrates at the surface of the liquid, and does not significantly affect sensitive biomolecules such as DNA. Finally electrospray has the ability to generate structured micro/nanoparticles in a more controlled way with high drug/nucleic acid encapsulation

Electrospray is a critical element of electrospray ionization mass-spectrometry, an analytical technique used to detect macromolecules that was developed by the 2002 chemistry Nobel Prize winner, Dr. John B. Fenn. (Fenn et al. 1989) Since then research has focused both on developing a fundamental understanding of the process as well as exploring potential applications of electrospray in fields ranging from the semiconductor industry to life science. During the past two decades, electrospray has been used to assist pyrolysis reactions and chemical vapor deposition processes, to produce inorganic particles including fine metal powder (Sn, Ag, Au, etc.), metal oxide particles (ZrO2, TiO2, etc.), ceramic particles (Si, SiO2), and semiconductor quantum dots (CdSe, GaAs, etc.) (Jaworek 2007; Salata 2005). Various structured particles have been produced via electrospray, including

that are particularly promising for advancing molecular and genetic medicine.

**1. Introduction**

efficiency.

**for Drug/Nucleic Acid Delivery**

*1NSF Nanoscale Science and Engineering Center, The Ohio State University, Columbus, Ohio*

*The Ohio State University, Columbus, Ohio*

## **Electrospray Production of Nanoparticles for Drug/Nucleic Acid Delivery**

Yun Wu1, Anthony Duong1,2, L. James Lee1,2 and Barbara E. Wyslouzil1,2,3 *1NSF Nanoscale Science and Engineering Center, The Ohio State University, Columbus, Ohio 2William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, Ohio 3Department of Chemistry, The Ohio State University, Columbus, Ohio USA*

### **1. Introduction**

222 The Delivery of Nanoparticles

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Nanomedicine – the application of nanotechnology to medicine – shows great potential to positively impact healthcare. Nanoparticles, including solid lipid nanoparticles, lipoplexes and polyplexes, can act as carriers to deliver the drugs or nucleic acid-based therapeutics that are particularly promising for advancing molecular and genetic medicine.

Many techniques have been developed to produce nanoparticles. Among them, electrospray has attracted recent research interest because it is an elegant and versatile way to make a broad array of nanoparticles. Electrospray is a technique that uses an electric field to disperse or break up a liquid. Compared with current technologies, such as bulk mixing, high pressure homogenization and double emulsion techniques, electrospray has three potential advantages. First, electrospray can generate monodisperse droplets whose size can vary from tens of nanometer to hundreds of micrometers, depending on the processing parameters. Secondly, it is a very gentle method. The free charge, induced by the electric field, only concentrates at the surface of the liquid, and does not significantly affect sensitive biomolecules such as DNA. Finally electrospray has the ability to generate structured micro/nanoparticles in a more controlled way with high drug/nucleic acid encapsulation efficiency.

Electrospray is a critical element of electrospray ionization mass-spectrometry, an analytical technique used to detect macromolecules that was developed by the 2002 chemistry Nobel Prize winner, Dr. John B. Fenn. (Fenn et al. 1989) Since then research has focused both on developing a fundamental understanding of the process as well as exploring potential applications of electrospray in fields ranging from the semiconductor industry to life science. During the past two decades, electrospray has been used to assist pyrolysis reactions and chemical vapor deposition processes, to produce inorganic particles including fine metal powder (Sn, Ag, Au, etc.), metal oxide particles (ZrO2, TiO2, etc.), ceramic particles (Si, SiO2), and semiconductor quantum dots (CdSe, GaAs, etc.) (Jaworek 2007; Salata 2005). Various structured particles have been produced via electrospray, including

Electrospray Production of Nanoparticles for Drug/Nucleic Acid Delivery 225

Taylor cone-jet and multi-jet mode. Taylor cone-jet mode is the most common used electrospray mode, because it can produce highly monodisperse particles in a stable

Fig. 1. Schematic diagrams of **(a)** standard electrospray **(b)** coaxial electrospray and **(c)** multiplexed electrospray. **(d)** Taylor cone-jet from standard electrospray **(e)** 91 nozzles used in multiplexed electrospray and **(f)** 91 Taylor cone-jets formed simultaneously.

Fig. 2. The electrospray modes change as the voltage increases. From left to right: dripping,

In cone-jet mode, the droplet size scales with the liquid flow rate, and is inversely proportional to the liquid conductivity. Theoretically droplet sizes can be determined from

the following scaling law, confirmed by many experiments (Jaworek 2007).

(Figures (e) and (f) are courtesy of Dr. Weiwei Deng).

microdripping, spindle, Taylor cone-jet and multi-jet.

manner.

poly(methyl methacrylate)-pigment nanoparticles (Widiyandari et al. 2007), cocoa butter microcapsules containing a sugar solution or an oil-in-water emulsion (Loscertales et al. 2002; Bocanegra et al. 2005) and microbubble suspensions (Farook et al. 2007), to name a few. Electrospray has been used to deposit particle suspensions to form thin film (Jaworek & Sobczyk 2008, Jaworek 2010) or on-demand patterns, such as silica particle coatings on a quartz glass (Jaworek 2007; Salata 2005). Electrospray has been successfully applied in tissue engineering, for example, polymer materials including poly(lactide-co-glycolide) or poly(ethylene glycol) were electrospray-coated on biomedical implants (Kumbar et al. 2007). Combined with electrospinning, electrospray was used to fabricate smooth muscle cell integrated blood vessel constructs. (Stankusa et al. 2007) In the area of drug/nucleic acid delivery, many biological materials, such as DNA, proteins, and lipids have been electrosprayed without changing their biological activity (Pareta et al. 2005; Jayasinghe et al. 2005; Davies et al. 2005; Wu et al. 2009a, 2009b, 2010, 2011). Proteins, such as bovine serum albumin, have been encapsulated in biodegradable polymeric microcapsules (Pareta and Edirisinghe, 2006; Xie & Wang, 2006), and small molecule drugs, such as taxol and griseofulvin, have been encapsulated in polymeric microparticles for systemic or oral delivery. (Xie et al. 2006; Zhang et al, 2011)

### **2. Principles of electrospray**

As illustrated in Figure 1a, in the standard electrospray configuration, a conducting liquid is slowly injected through a needle by a syringe pump. An electrical potential is applied to the needle to introduce free charge at the liquid surface. The free charge generates electric stress that causes the liquid to accelerate away from the needle. When the electrical potential rises to several kilovolts, the liquid meniscus at the needle opening develops into a conical shape, commonly called the Taylor cone. At the cone apex, where the free charge is highly concentrated, a liquid jet with high charge density is observed. Monodisperse particles are formed when the jet breaks into fine particles due to varicose or kink instabilities. (Cloupeau et al. 1994; Jaworek 2007; Salata 2005; Loscertales et al. 2002). Typically, the initial micron size droplets contain both solvent and a non-volatile solute and nanoparticles are produced as solvent evaporates from the high surface area aerosol.

Other electrospray configurations are also possible. Figure 1(b) shows a coaxial electrospray configuration, where two liquids are fed through the inner needle and the outer needle, respectively. This configuration is widely used to produce structured nanoparticles. Finally, since the flow rate in a single electrospray setup is always low (uL/hr or mL/hr), multiplexed electrospray configurations have been developed to scale up production. (Deng et al, 2006) As shown in Figure 1(c), the single needle is replaced by a micro-nozzle array. All nozzles work simultaneously and produce nanoparticles with the liquid flow increased by orders of magnitude.

The electrospray modes and resulting droplet sizes *d*D are controlled by the process parameters that include the applied voltage V, liquid flow rate Q, and liquid properties including electrical conductivity , surface tension , and liquid density . (Cloupeau et al. 1994; Jaworek 2007; Salata 2005; Loscertales et al. 2002, Ganan-Calvo AM. 2004; Hartman et al. 1999; Basak et al. 2007) As illustrated in Figure 2, different electrospray modes are obtained as the applied voltage increases. (Cloupeau et al. 1994; Jaworek and Krupa 1999; Chen et al. 2005; Yurteri et al. 2010) These include dripping, micro-dripping, spindle,

poly(methyl methacrylate)-pigment nanoparticles (Widiyandari et al. 2007), cocoa butter microcapsules containing a sugar solution or an oil-in-water emulsion (Loscertales et al. 2002; Bocanegra et al. 2005) and microbubble suspensions (Farook et al. 2007), to name a few. Electrospray has been used to deposit particle suspensions to form thin film (Jaworek & Sobczyk 2008, Jaworek 2010) or on-demand patterns, such as silica particle coatings on a quartz glass (Jaworek 2007; Salata 2005). Electrospray has been successfully applied in tissue engineering, for example, polymer materials including poly(lactide-co-glycolide) or poly(ethylene glycol) were electrospray-coated on biomedical implants (Kumbar et al. 2007). Combined with electrospinning, electrospray was used to fabricate smooth muscle cell integrated blood vessel constructs. (Stankusa et al. 2007) In the area of drug/nucleic acid delivery, many biological materials, such as DNA, proteins, and lipids have been electrosprayed without changing their biological activity (Pareta et al. 2005; Jayasinghe et al. 2005; Davies et al. 2005; Wu et al. 2009a, 2009b, 2010, 2011). Proteins, such as bovine serum albumin, have been encapsulated in biodegradable polymeric microcapsules (Pareta and Edirisinghe, 2006; Xie & Wang, 2006), and small molecule drugs, such as taxol and griseofulvin, have been encapsulated in polymeric microparticles for systemic or oral

As illustrated in Figure 1a, in the standard electrospray configuration, a conducting liquid is slowly injected through a needle by a syringe pump. An electrical potential is applied to the needle to introduce free charge at the liquid surface. The free charge generates electric stress that causes the liquid to accelerate away from the needle. When the electrical potential rises to several kilovolts, the liquid meniscus at the needle opening develops into a conical shape, commonly called the Taylor cone. At the cone apex, where the free charge is highly concentrated, a liquid jet with high charge density is observed. Monodisperse particles are formed when the jet breaks into fine particles due to varicose or kink instabilities. (Cloupeau et al. 1994; Jaworek 2007; Salata 2005; Loscertales et al. 2002). Typically, the initial micron size droplets contain both solvent and a non-volatile solute and nanoparticles are produced

Other electrospray configurations are also possible. Figure 1(b) shows a coaxial electrospray configuration, where two liquids are fed through the inner needle and the outer needle, respectively. This configuration is widely used to produce structured nanoparticles. Finally, since the flow rate in a single electrospray setup is always low (uL/hr or mL/hr), multiplexed electrospray configurations have been developed to scale up production. (Deng et al, 2006) As shown in Figure 1(c), the single needle is replaced by a micro-nozzle array. All nozzles work simultaneously and produce nanoparticles with the liquid flow increased

The electrospray modes and resulting droplet sizes *d*D are controlled by the process parameters that include the applied voltage V, liquid flow rate Q, and liquid properties including electrical conductivity , surface tension , and liquid density . (Cloupeau et al. 1994; Jaworek 2007; Salata 2005; Loscertales et al. 2002, Ganan-Calvo AM. 2004; Hartman et al. 1999; Basak et al. 2007) As illustrated in Figure 2, different electrospray modes are obtained as the applied voltage increases. (Cloupeau et al. 1994; Jaworek and Krupa 1999; Chen et al. 2005; Yurteri et al. 2010) These include dripping, micro-dripping, spindle,

delivery. (Xie et al. 2006; Zhang et al, 2011)

as solvent evaporates from the high surface area aerosol.

**2. Principles of electrospray**

by orders of magnitude.

Taylor cone-jet and multi-jet mode. Taylor cone-jet mode is the most common used electrospray mode, because it can produce highly monodisperse particles in a stable manner.

Fig. 1. Schematic diagrams of **(a)** standard electrospray **(b)** coaxial electrospray and **(c)** multiplexed electrospray. **(d)** Taylor cone-jet from standard electrospray **(e)** 91 nozzles used in multiplexed electrospray and **(f)** 91 Taylor cone-jets formed simultaneously. (Figures (e) and (f) are courtesy of Dr. Weiwei Deng).

Fig. 2. The electrospray modes change as the voltage increases. From left to right: dripping, microdripping, spindle, Taylor cone-jet and multi-jet.

In cone-jet mode, the droplet size scales with the liquid flow rate, and is inversely proportional to the liquid conductivity. Theoretically droplet sizes can be determined from the following scaling law, confirmed by many experiments (Jaworek 2007).

Electrospray Production of Nanoparticles for Drug/Nucleic Acid Delivery 227

The standard electrospray configuration (Figure 1a) was used to produce cholesterol nanoparticles dispersed in aqueous media. Cholesterol powder (Sigma C3045) was dissolved in ethanol (Pharmco-Aaper E200GP) at a concentration of 2 mg/mL and then delivered to a 27 gauge needle by a syringe pump at a flow rate of 2 mL/h. A voltage of approximate 2.5 kV was applied to the needle and the observation of a stable Taylor cone-jet mode confirmed the proper conditions for generating fine droplets. The ethanol evaporated from the droplets, aided by heat input from a lamp, and the residual cholesterol particles were captured in 1X PBS supplemented with 1% Pluronic F68 placed in a grounded aluminum dish 5.5 cm below the needle tip. Figure 3 illustrates a typical size distribution measured for the cholesterol nanoparticle suspension. Highly monodisperse cholesterol nanoparticles were produced with sizes ~150 nm. The cholesterol concentration in the final solution was 180+10 g/mL prior to sterile filtration and 150+8 g/mL afterwards, i.e. up to

**3.1.2 Production of cholesterol nanoparticles**

100 times higher than the solubility limit for cholesterol.

100

80

60

Volume

v was 140 nm and polydispersity was 0.107.

electrospray.

40

20

0

**3.1.3 Bioavailability of cholesterol nanoparticles for NS0 cells**

5 6 7 8 9

Fig. 3. A typical size distribution of cholesterol nanoparticles, in which Dp, mean,

<sup>100</sup> <sup>2</sup> <sup>3</sup> <sup>4</sup> <sup>5</sup>

Cholesterol

Dp, nm

In this study, cholesterol itself was the bio-active ingredient, and NS0 cells were used to investigate the bioavailability of the cholesterol nanoparticles. While NS0 cells normally require serum as a cholesterol source, several research groups have demonstrated the growth of NS0 cells in protein free and chemically defined medium, using chemically defined cholesterol supplements. (Zhang & Robinson, 2005; Talley et al. 2005; Ojito et al. 2001) The auxotrophic nature of NS0 cells with respect to cholesterol makes this cell line a natural choice for characterizing the bioavailability of cholesterol nanoparticles produced by

NS0 cells were continuously cultured in 250 mL shake flasks with 50 mL working volume in the basal medium supplemented with cholesterol nanoparticles or SyntheChol NS0 supplement (Sigma S5442, SyntheChol for short) at a cholesterol concentration level of 3.5 g/mL. SyntheChol is a proprietary cholesterol supplement in liquid form and acts as the positive control. Figure 4 illustrates the viable cell density and cell viability as a function of

$$d\_D = a \left(\frac{\mathbb{Q}^3 \varepsilon\_0 \rho}{\pi^4 \sigma \gamma}\right)^{1/6} (\alpha = 2.9)$$

where 0 is the vacuum permittivity. In general, droplet size decreases with decreasing flow rate and increasing electrical conductivity of the liquid. By adjusting the flow rate and the liquid properties, droplets with the desired size can be produced in a well-controlled manner. Frequently, the initial solution contains both solvent and solute, and the final particle size ( *<sup>p</sup> d* ) is related to the initial droplet size *d*D by the following equation:

$$d\_p = d\_D \left(\frac{c\rho}{\rho\_p}\right)^{1/3}$$

where c is the concentration of solute and p is the density of the particle.

### **3. Electrospray production of nanoparticles for drug/nucleic acid delivery**

Our research group has been the first to explore electrospray as a means to produce solid lipid nanoparticles, lipoplexes and polyplexes for drug/nucleic acid delivery. Our results demonstrate the great potential of electrospray to produce nanoparticles in a well-controlled manner, and opens new avenues for the development of nanomedicine.

#### **3.1 Solid lipid nanoparticles for hydrophobic drug delivery**

#### **3.1.1 Introduction**

Solid lipid nanoparticles (SLNs) have been proposed as an alternative drug delivery system to traditional colloidal carriers, such as emulsions, liposomes and polymeric nanoparticles. As drug delivery vehicles, emulsions and liposomes are often limited by physical instability and low drug loading capacity. (Müller et al. 2000, 2004) Polymeric nanoparticles can exhibit cytotoxicity, and there are no industrialized production methods. SLNs may provide a drug delivery system that avoids many of these problems, and high pressure homogenization (HPH), the most popular preparation technique, is a reasonable method for large-scale production. Unfortunately, HPH requires molten lipids and high energy input. Thus, it is often unsuitable for use with temperature and shear sensitive biomolecules. Furthermore, as the mixtures cool, some drugs do not incorporate into the lipids but stick on the surface, resulting in burst drug release kinetics and low drug loading capacity. (Müller et al. 2000, 2004)

In our work we used electrospray to produce solid lipid nanoparticles. (Wu et al. 2009a, 2011) Cholesterol was chosen as a model since it is not only an important lipid but also a good model for lipophilic drugs. Similar to most lipophilic drugs, cholesterol has very low solubility in aqueous solutions, on the order of 1.8g/mL or 4.7M. (Haberland & Reynolds, 1973) Cholesterol has similar structure as the well-known anticancer drug Taxol®, and cholesterol (molecular weight = 387.6) is also a small molecule. In addition, the solubility of lipophilic drugs in lipids is much higher than in aqueous solutions, and, therefore, lipid nanoparticles, like the cholesterol nanoparticles discussed here, may also be useful as carriers to deliver lipophilic drugs.

#### **3.1.2 Production of cholesterol nanoparticles**

226 The Delivery of Nanoparticles

1/6 <sup>3</sup> 0

*<sup>Q</sup> <sup>d</sup>* (=2.9)

where 0 is the vacuum permittivity. In general, droplet size decreases with decreasing flow rate and increasing electrical conductivity of the liquid. By adjusting the flow rate and the liquid properties, droplets with the desired size can be produced in a well-controlled manner. Frequently, the initial solution contains both solvent and solute, and the final

*p*

1/3

*D* 4

particle size ( *<sup>p</sup> d* ) is related to the initial droplet size *d*D by the following equation:

*p D*

where c is the concentration of solute and p is the density of the particle.

manner, and opens new avenues for the development of nanomedicine.

**3.1 Solid lipid nanoparticles for hydrophobic drug delivery**

**3.1.1 Introduction**

2004)

carriers to deliver lipophilic drugs.

*<sup>c</sup> d d*

**3. Electrospray production of nanoparticles for drug/nucleic acid delivery**

Our research group has been the first to explore electrospray as a means to produce solid lipid nanoparticles, lipoplexes and polyplexes for drug/nucleic acid delivery. Our results demonstrate the great potential of electrospray to produce nanoparticles in a well-controlled

Solid lipid nanoparticles (SLNs) have been proposed as an alternative drug delivery system to traditional colloidal carriers, such as emulsions, liposomes and polymeric nanoparticles. As drug delivery vehicles, emulsions and liposomes are often limited by physical instability and low drug loading capacity. (Müller et al. 2000, 2004) Polymeric nanoparticles can exhibit cytotoxicity, and there are no industrialized production methods. SLNs may provide a drug delivery system that avoids many of these problems, and high pressure homogenization (HPH), the most popular preparation technique, is a reasonable method for large-scale production. Unfortunately, HPH requires molten lipids and high energy input. Thus, it is often unsuitable for use with temperature and shear sensitive biomolecules. Furthermore, as the mixtures cool, some drugs do not incorporate into the lipids but stick on the surface, resulting in burst drug release kinetics and low drug loading capacity. (Müller et al. 2000,

In our work we used electrospray to produce solid lipid nanoparticles. (Wu et al. 2009a, 2011) Cholesterol was chosen as a model since it is not only an important lipid but also a good model for lipophilic drugs. Similar to most lipophilic drugs, cholesterol has very low solubility in aqueous solutions, on the order of 1.8g/mL or 4.7M. (Haberland & Reynolds, 1973) Cholesterol has similar structure as the well-known anticancer drug Taxol®, and cholesterol (molecular weight = 387.6) is also a small molecule. In addition, the solubility of lipophilic drugs in lipids is much higher than in aqueous solutions, and, therefore, lipid nanoparticles, like the cholesterol nanoparticles discussed here, may also be useful as The standard electrospray configuration (Figure 1a) was used to produce cholesterol nanoparticles dispersed in aqueous media. Cholesterol powder (Sigma C3045) was dissolved in ethanol (Pharmco-Aaper E200GP) at a concentration of 2 mg/mL and then delivered to a 27 gauge needle by a syringe pump at a flow rate of 2 mL/h. A voltage of approximate 2.5 kV was applied to the needle and the observation of a stable Taylor cone-jet mode confirmed the proper conditions for generating fine droplets. The ethanol evaporated from the droplets, aided by heat input from a lamp, and the residual cholesterol particles were captured in 1X PBS supplemented with 1% Pluronic F68 placed in a grounded aluminum dish 5.5 cm below the needle tip. Figure 3 illustrates a typical size distribution measured for the cholesterol nanoparticle suspension. Highly monodisperse cholesterol nanoparticles were produced with sizes ~150 nm. The cholesterol concentration in the final solution was 180+10 g/mL prior to sterile filtration and 150+8 g/mL afterwards, i.e. up to 100 times higher than the solubility limit for cholesterol.

Fig. 3. A typical size distribution of cholesterol nanoparticles, in which Dp, mean, v was 140 nm and polydispersity was 0.107.

#### **3.1.3 Bioavailability of cholesterol nanoparticles for NS0 cells**

In this study, cholesterol itself was the bio-active ingredient, and NS0 cells were used to investigate the bioavailability of the cholesterol nanoparticles. While NS0 cells normally require serum as a cholesterol source, several research groups have demonstrated the growth of NS0 cells in protein free and chemically defined medium, using chemically defined cholesterol supplements. (Zhang & Robinson, 2005; Talley et al. 2005; Ojito et al. 2001) The auxotrophic nature of NS0 cells with respect to cholesterol makes this cell line a natural choice for characterizing the bioavailability of cholesterol nanoparticles produced by electrospray.

NS0 cells were continuously cultured in 250 mL shake flasks with 50 mL working volume in the basal medium supplemented with cholesterol nanoparticles or SyntheChol NS0 supplement (Sigma S5442, SyntheChol for short) at a cholesterol concentration level of 3.5 g/mL. SyntheChol is a proprietary cholesterol supplement in liquid form and acts as the positive control. Figure 4 illustrates the viable cell density and cell viability as a function of

Electrospray Production of Nanoparticles for Drug/Nucleic Acid Delivery 229

The NS0 cell line is one of the important cell lines widely used by the pharmaceutical industry to produce therapeutic antibodies. We therefore investigated the effect of the cholesterol source on product titer by conducting a fed-batch culture of NS0 cells using electrosprayed cholesterol nanoparticles and the SyntheChol supplement at feed rates of 0.7, 1.75 and 3.5 g/mL/day. Figure 5 summarizes the viable cell density, cell viability and normalized product titer (relative to the 1.75 g/mL/day SyntheChol supplement). At the lowest feed rate of 0.7 g/mL/day, neither cholesterol nanoparticles nor SyntheChol supplement provided sufficient cholesterol to the cells and so cell growth was limited and the corresponding product titer was low (Figure 5c). When the feed rate of SyntheChol was increased, cell growth improved up to a feed rate of 1.75 g/mL/day but was compromised at feed rate of 3.5 g/mL/day. The inhibitory effect of 3.5 g/mL/day feeding was presumably due to the compounds in SyntheChol that solubilize the cholesterol rather than to the presence of cholesterol. For cholesterol nanoparticles, both cell growth and viability improved at feed rates higher than 0.7 g/mL/day. Compared to SyntheChol, culturing was extended from 8 days to 10 days. In general, the performance of cholesterol nanoparticles was better than that of SyntheChol supplementation at the same cholesterol feed rates. For both cholesterol nanoparticles and SyntheChol supplements, the highest product titer was achieved at a feed rate of 1.75 g/mL/day and the product titer with

Compared with conventional technologies, we have demonstrated that electrospray is an efficient way to produce solid lipid nanoparticles for drug delivery. In our application we used electrospray to produce highly monodisperse cholesterol nanoparticles, and investigated the bioavailability of the nanoparticles using the cholesterol auxotrophic NS0 cell line with SyntheChol as the positive control. We found that the cholesterol nanoparticles not only supported NS0 cell growth but also improved the titer of therapeutic antibody by

Viral and non-viral vectors have been developed to deliver nucleic acids to treat genetic and acquired disease. (Pack et al., 2005; Mastrobattista et al., 2006) Compared to viral vectors, non-vrial vectors show lower immunogenicity, lower toxicity, better stability and lower cost. (Laporte et al., 2006; Kircheis, et al., 2001; Gebhart and Kabanov, 2001). Among the nonviral vectors, polyplexes and lipoplexes represent two major carrier systems. In this section, we focus on polyplexes for gene delivery, while in the following section we discuss the use of

Polyethylenimines (PEI) is a cationic polymer widely used to condense DNA and form polyplexes. Although PEI shows great promise for gene delivery both *in vitro* and *in vivo*  (Godbey, et al., 1999; Neu et al., 2005), the delivery efficiency highly depends on the N/P ratio (the molar ratio of nitrogen in PEI to phosphate in DNA) and the preparation method. Bulk mixing (BM), the most commonly used method, is a simple and straightforward way to prepare DNA/PEI polyplexes. Unfortunately, the formation of the polyplexes is not well

cholesterol nanoparticles was ~32% higher than that with SyntheChol.

**3.1.4 Conclusions**

**3.2.1 Introduction**

32% when compared with SyntheChol.

lipoplexes for oligonucleotides delivery.

**3.2 DNA/polycation polyplexes for gene delivery**

time. NS0 cells grew better with cholesterol nanoparticles than with SyntheChol reaching much higher peak cell densities: 5.28e6 cells/mL vs. 3.35e6 cells/mL.

Fig. 4. Growth of NS0 cells in media supplemented with 3.5 g/mL of cholesterol nanoparticles or SyntheChol: viable cell density **(a)** and cell viability **(b)**. The results are averages of triplicate runs.

Fig. 5. The effect of cholesterol concentration on **(a)** the growth, **(b)** the viability, and **(c)** the product titer of NS0 cells during fed-batch culture. Cholesterol nanoparticles or SyntheChol supplement were fed on day 0 and daily from day 2 till the end of culture at cholesterol concentration levels of 0.7 g/mL, 1.75 g/mL or 3.5 g/mL. Error bars indicate one standard deviation of the results of triplicate runs. (ES CN: electrosprayed cholesterol nanoparticles)

The NS0 cell line is one of the important cell lines widely used by the pharmaceutical industry to produce therapeutic antibodies. We therefore investigated the effect of the cholesterol source on product titer by conducting a fed-batch culture of NS0 cells using electrosprayed cholesterol nanoparticles and the SyntheChol supplement at feed rates of 0.7, 1.75 and 3.5 g/mL/day. Figure 5 summarizes the viable cell density, cell viability and normalized product titer (relative to the 1.75 g/mL/day SyntheChol supplement). At the lowest feed rate of 0.7 g/mL/day, neither cholesterol nanoparticles nor SyntheChol supplement provided sufficient cholesterol to the cells and so cell growth was limited and the corresponding product titer was low (Figure 5c). When the feed rate of SyntheChol was increased, cell growth improved up to a feed rate of 1.75 g/mL/day but was compromised at feed rate of 3.5 g/mL/day. The inhibitory effect of 3.5 g/mL/day feeding was presumably due to the compounds in SyntheChol that solubilize the cholesterol rather than to the presence of cholesterol. For cholesterol nanoparticles, both cell growth and viability improved at feed rates higher than 0.7 g/mL/day. Compared to SyntheChol, culturing was extended from 8 days to 10 days. In general, the performance of cholesterol nanoparticles was better than that of SyntheChol supplementation at the same cholesterol feed rates. For both cholesterol nanoparticles and SyntheChol supplements, the highest product titer was achieved at a feed rate of 1.75 g/mL/day and the product titer with cholesterol nanoparticles was ~32% higher than that with SyntheChol.

### **3.1.4 Conclusions**

228 The Delivery of Nanoparticles

time. NS0 cells grew better with cholesterol nanoparticles than with SyntheChol reaching

much higher peak cell densities: 5.28e6 cells/mL vs. 3.35e6 cells/mL.

Fig. 4. Growth of NS0 cells in media supplemented with 3.5 g/mL of cholesterol nanoparticles or SyntheChol: viable cell density **(a)** and cell viability **(b)**. The results are

Fig. 5. The effect of cholesterol concentration on **(a)** the growth, **(b)** the viability, and **(c)** the product titer of NS0 cells during fed-batch culture. Cholesterol nanoparticles or SyntheChol supplement were fed on day 0 and daily from day 2 till the end of culture at cholesterol concentration levels of 0.7 g/mL, 1.75 g/mL or 3.5 g/mL. Error bars indicate one standard deviation of the results of triplicate runs. (ES CN: electrosprayed cholesterol

averages of triplicate runs.

nanoparticles)

Compared with conventional technologies, we have demonstrated that electrospray is an efficient way to produce solid lipid nanoparticles for drug delivery. In our application we used electrospray to produce highly monodisperse cholesterol nanoparticles, and investigated the bioavailability of the nanoparticles using the cholesterol auxotrophic NS0 cell line with SyntheChol as the positive control. We found that the cholesterol nanoparticles not only supported NS0 cell growth but also improved the titer of therapeutic antibody by 32% when compared with SyntheChol.

### **3.2 DNA/polycation polyplexes for gene delivery**

#### **3.2.1 Introduction**

Viral and non-viral vectors have been developed to deliver nucleic acids to treat genetic and acquired disease. (Pack et al., 2005; Mastrobattista et al., 2006) Compared to viral vectors, non-vrial vectors show lower immunogenicity, lower toxicity, better stability and lower cost. (Laporte et al., 2006; Kircheis, et al., 2001; Gebhart and Kabanov, 2001). Among the nonviral vectors, polyplexes and lipoplexes represent two major carrier systems. In this section, we focus on polyplexes for gene delivery, while in the following section we discuss the use of lipoplexes for oligonucleotides delivery.

Polyethylenimines (PEI) is a cationic polymer widely used to condense DNA and form polyplexes. Although PEI shows great promise for gene delivery both *in vitro* and *in vivo*  (Godbey, et al., 1999; Neu et al., 2005), the delivery efficiency highly depends on the N/P ratio (the molar ratio of nitrogen in PEI to phosphate in DNA) and the preparation method. Bulk mixing (BM), the most commonly used method, is a simple and straightforward way to prepare DNA/PEI polyplexes. Unfortunately, the formation of the polyplexes is not well

Electrospray Production of Nanoparticles for Drug/Nucleic Acid Delivery 231

observations are consistent with those reported by Davies et al. (2005) and confirm that

Fig. 6. Gel electrophoresis shows that the pDNA is not damaged during the electrospray process. M: marker lane. C: control. E: electrosprayed pDNAs. Electrospray conditions: pGFP: flowrate of 6 mL/h and voltage of 5.0 kV; pSEAP: flowrate of 6 mL/h and voltage of

Fig. 7. Typical size distributions of the pDNA/PEI polyplexes prepared by coaxial electrospray and bulk mixing. The flow rate was 6 mL/h and the voltage was 5.0 kV for

pGFP/PEI polyplexes and 5.6 kV for pSEAP/PEI polyplexes.

5.6 kV. OC: open circular. SC: supercoiled plasmid.

coaxial electrospray is a gentle but effective way to spray delicate bio-molecules.

controlled in bulk mixing, significantly affecting the particle size, structure, and, thus, the delivery efficiency. (Kircheis, et al., 2001; Gebhart and Kabanov, 2001) Alternative methods are needed to overcome this challenge.

Because electrospray is a gentle method that does not damage biomolecules it has attracted research interest in the realm of gene delivery. In some of the earliest work, enhanced green fluorescent protein (eGFP) plasmid was successfully delivered into African Green Monkey fibroblast cells (COS-1) using coaxial electrospray. (Chen et al. 2000) Since electrospray generates an aerosol, Davies et al. (2005) investigated gene delivery by exposing mice to naked pCIKLux aerosol generated via electrospray. Unfortunately, the *in vivo* delivery efficiency, 0.075%, was much lower than the 0.2% delivery efficiency achieved by Koshkina et al. (2003) when they nebulized pDNA/PEI polyplexes for pulmonary gene delivery. One possible reason for the low delivery efficiency of electrosprayed pCIKLux might be that the naked pDNA used is not stable in biological fluids and does not interact with cell membrane due to its negative charges. Our hypothesis was that pDNA/PEI polyplexes produced via electrospray might overcome limitations in the earlier studies. Thus, we used coaxial electrospray to achieve better control over the mixing of the DNA solution and the PEI solution, and thus to produce DNA/PEI polyplexes in a well-controlled manner. (Wu et al. 2010)

### **3.2.2 Coaxial electrospray DNA/PEI polyplexes**

Coaxial electrospray was explored as a method to produce pDNA/PEI polyplexes using pGFP and pSEAP for qualitative and quantitative studies, respectively. The pDNAs were dispersed in OPTI-MEM medium at a concentration of 20 µg/mL. Branched 25 kDa PEI was dissolved in OPTI-MEM medium at concentrations of 17.2 µg/mL. In the coaxial electrospray setup, the pDNA solution flowed through the inner 27 gauge needle and the PEI solution flowed through the outer 20 gauge needle. The flow rates of the pDNA and PEI solutions were both set at 6mL/hr to yield an N/P ratio of 6.7. A positive voltage, typically 5~6 kV, was applied between the inner needle and a grounded copper ring electrode. The polyplexes were produced in the stable Taylor cone jet mode, captured in a grounded aluminum dish 5 cm below the needle tip, and used within 10 minutes.

The conventional bulk mixing method was also used to prepare pDNA/PEI polyplexes. Here PEI solution (17.2 µg/mL) was added into equal volume pDNA (20 µg/mL) solution to achieve an N/P ratio of 6.7. The resulting mixture was vortexed for a few seconds, incubated at room temperature for 10 minutes and used immediately.

### **3.2.3 pDNA damage analysis**

To investigate the effects of coaxial electrospray on pDNA integrity, pGFP and pSEAP solutions were coaxial electrosprayed with OPTI-MEM medium containing no PEI. The flow rate for both pDNAs was set at 6mL/h, and the voltages were set at 5.0 kV for pGFP and 5.6 kV for pSEAP. Agarose gel electrophoresis was used to detect if the pDNA was damaged by coaxial electrospray. As illustrated in Figure 6, compared to the non-electrosprayed pDNA controls, little degradation of pDNA is observed. In addition, the ratio of supercoiled to open circular pDNA of the sprayed samples appears unchanged relative to the controls. Our

controlled in bulk mixing, significantly affecting the particle size, structure, and, thus, the delivery efficiency. (Kircheis, et al., 2001; Gebhart and Kabanov, 2001) Alternative methods

Because electrospray is a gentle method that does not damage biomolecules it has attracted research interest in the realm of gene delivery. In some of the earliest work, enhanced green fluorescent protein (eGFP) plasmid was successfully delivered into African Green Monkey fibroblast cells (COS-1) using coaxial electrospray. (Chen et al. 2000) Since electrospray generates an aerosol, Davies et al. (2005) investigated gene delivery by exposing mice to naked pCIKLux aerosol generated via electrospray. Unfortunately, the *in vivo* delivery efficiency, 0.075%, was much lower than the 0.2% delivery efficiency achieved by Koshkina et al. (2003) when they nebulized pDNA/PEI polyplexes for pulmonary gene delivery. One possible reason for the low delivery efficiency of electrosprayed pCIKLux might be that the naked pDNA used is not stable in biological fluids and does not interact with cell membrane due to its negative charges. Our hypothesis was that pDNA/PEI polyplexes produced via electrospray might overcome limitations in the earlier studies. Thus, we used coaxial electrospray to achieve better control over the mixing of the DNA solution and the PEI solution, and thus to

produce DNA/PEI polyplexes in a well-controlled manner. (Wu et al. 2010)

aluminum dish 5 cm below the needle tip, and used within 10 minutes.

incubated at room temperature for 10 minutes and used immediately.

Coaxial electrospray was explored as a method to produce pDNA/PEI polyplexes using pGFP and pSEAP for qualitative and quantitative studies, respectively. The pDNAs were dispersed in OPTI-MEM medium at a concentration of 20 µg/mL. Branched 25 kDa PEI was dissolved in OPTI-MEM medium at concentrations of 17.2 µg/mL. In the coaxial electrospray setup, the pDNA solution flowed through the inner 27 gauge needle and the PEI solution flowed through the outer 20 gauge needle. The flow rates of the pDNA and PEI solutions were both set at 6mL/hr to yield an N/P ratio of 6.7. A positive voltage, typically 5~6 kV, was applied between the inner needle and a grounded copper ring electrode. The polyplexes were produced in the stable Taylor cone jet mode, captured in a grounded

The conventional bulk mixing method was also used to prepare pDNA/PEI polyplexes. Here PEI solution (17.2 µg/mL) was added into equal volume pDNA (20 µg/mL) solution to achieve an N/P ratio of 6.7. The resulting mixture was vortexed for a few seconds,

To investigate the effects of coaxial electrospray on pDNA integrity, pGFP and pSEAP solutions were coaxial electrosprayed with OPTI-MEM medium containing no PEI. The flow rate for both pDNAs was set at 6mL/h, and the voltages were set at 5.0 kV for pGFP and 5.6 kV for pSEAP. Agarose gel electrophoresis was used to detect if the pDNA was damaged by coaxial electrospray. As illustrated in Figure 6, compared to the non-electrosprayed pDNA controls, little degradation of pDNA is observed. In addition, the ratio of supercoiled to open circular pDNA of the sprayed samples appears unchanged relative to the controls. Our

**3.2.2 Coaxial electrospray DNA/PEI polyplexes**

**3.2.3 pDNA damage analysis**

are needed to overcome this challenge.

observations are consistent with those reported by Davies et al. (2005) and confirm that coaxial electrospray is a gentle but effective way to spray delicate bio-molecules.

Fig. 6. Gel electrophoresis shows that the pDNA is not damaged during the electrospray process. M: marker lane. C: control. E: electrosprayed pDNAs. Electrospray conditions: pGFP: flowrate of 6 mL/h and voltage of 5.0 kV; pSEAP: flowrate of 6 mL/h and voltage of 5.6 kV. OC: open circular. SC: supercoiled plasmid.

Fig. 7. Typical size distributions of the pDNA/PEI polyplexes prepared by coaxial electrospray and bulk mixing. The flow rate was 6 mL/h and the voltage was 5.0 kV for pGFP/PEI polyplexes and 5.6 kV for pSEAP/PEI polyplexes.

Electrospray Production of Nanoparticles for Drug/Nucleic Acid Delivery 233

electrospray is an aerosol technique, it should be a useful way to deliver pDNA/PEI polyplexes directly to the lungs and achieve higher gene delivery efficiency than either electrospraying naked pDNA or nebulizing pDNA/PEI polyplexes produced by

Fig. 8. **(a)** The GFP expression in NIH 3T3 cells and **(b)** the cell viability measured 2 days post transfection. All polyplexes were produced at the N/P ratio of 6.7. For coaxial

Fig. 9. The pSEAP expression in NIH 3T3 **(a)** and the cell viability **(b)** measured 2 days post transfection. All polyplexes were produced at the N/P ratio of 6.7. For coaxial electrospray,

electrospray, the flow rate was 6mL/h and voltage was 5.0kV. (n=4)

the flow rate was 6 mL/h and voltage was 5.6 kV. (n=5, \*\*: p<0.005)

conventional methods.

### **3.2.4 Size distribution of pDNA/PEI polyplexes**

Dynamic light scattering was used to measure the size distribution of pGFP/PEI and pSEAP/PEI polyplexes prepared by coaxial electrospray, and bulk mixing. (Figure 7) The pDNA/PEI polyplexes prepared by either method are relatively monodisperse. The pGFP/PEI polyplexes prepared by coaxial electrospray had smaller particle size compared to those prepared by bulk mixing, while the sizes of the pSEAP/PEI polyplexes prepared by coaxial electrospray and bulk mixing were comparable. We note that the polyplexes prepared in this work were almost always larger than 400 nm because OPTI-MEM medium rather than NaCl solutions were used. OPTI-MEM medium was chosen to prepare polyplexes because it better mimics the *in vivo* situation than NaCl or PBS, and because its physical properties (electric conductivity and surface tension) are appropriate for the electrospray process. The downside is that OPTI-MEM medium contains reduced serum, and the adsorption of serum albumin and other negatively charged proteins enhances aggregation of polyplexes (Pack et al., 2005). Although the size distribution of each sample was measured within 10 minutes after preparation, the polyplexes were always larger than 300 nm suggesting that aggregates already formed. The adsorption of proteins to the polyplexes was also demonstrated by zeta potential measurements. The zeta potentials of all polyplexes samples were either close to zero or only slightly positive. Approaches such as grafting hydrophilic polymers, including polyethylene glycol (PEG) or polysaccharides, onto the PEI may help improve the stability of the polyplexes in serum (Pack et al., 2005; Laporte et al., 2006; Neu et al., 2001) and is one approach that could reduce aggregation.

### **3.2.5 pDNA/PEI polyplex transfection, DNA expression, and cell viability**

The delivery efficiency of pGFP/PEI polyplexes produced by coaxial electrospray and bulk mixing was evaluated in NIH 3T3 cells and compared to LipofectamineTM 2000, the positive control. As illustrated in Figure 8(a), at N/P ratio of 6.7, cells transfected with polyplexes produced by coaxial electrospray showed similar GFP expression as those transfected with LipofectamineTM 2000, while cells transfected with polyplexes produced by bulk mixing showed much less GFP expression. No significant difference in cell viability was found among coaxial electrospray, BM and LipofectamineTM 2000 (p<0.05).

Since the evaluation of GFP expression is only qualitative, pSEAP was used to compare the delivery efficiency of polyplexes in a more quantitative way. As shown in Figure 9 (a), at N/P ratio of 6.7, cells transfected with the pSEAP/PEI polyplexes produced by coaxial electrospray gave 2.6 times higher SEAP expression than those produced by bulk mixing. Although cells transfected with LipofectamineTM 2000 showed the highest SEAP expression, 3.3 times higher than bulk mixing, the cell viability was low because of the toxicity of this material. (Figure 9(b))

#### **3.2.6 Conclusions**

Both pGFP/PEI and pSEAP/PEI polyplexes were successfully produced using coaxial electrospray. The delivery efficiency of pDNA/PEI polyplexes was evalulated in NIH 3T3 cells. At N/P ratio of 6.7, polyplexes produced by coaxial electrospray were more effective at delivering genes to NIH 3T3 cells than those prepared by bulk mixing. Since coaxial

Dynamic light scattering was used to measure the size distribution of pGFP/PEI and pSEAP/PEI polyplexes prepared by coaxial electrospray, and bulk mixing. (Figure 7) The pDNA/PEI polyplexes prepared by either method are relatively monodisperse. The pGFP/PEI polyplexes prepared by coaxial electrospray had smaller particle size compared to those prepared by bulk mixing, while the sizes of the pSEAP/PEI polyplexes prepared by coaxial electrospray and bulk mixing were comparable. We note that the polyplexes prepared in this work were almost always larger than 400 nm because OPTI-MEM medium rather than NaCl solutions were used. OPTI-MEM medium was chosen to prepare polyplexes because it better mimics the *in vivo* situation than NaCl or PBS, and because its physical properties (electric conductivity and surface tension) are appropriate for the electrospray process. The downside is that OPTI-MEM medium contains reduced serum, and the adsorption of serum albumin and other negatively charged proteins enhances aggregation of polyplexes (Pack et al., 2005). Although the size distribution of each sample was measured within 10 minutes after preparation, the polyplexes were always larger than 300 nm suggesting that aggregates already formed. The adsorption of proteins to the polyplexes was also demonstrated by zeta potential measurements. The zeta potentials of all polyplexes samples were either close to zero or only slightly positive. Approaches such as grafting hydrophilic polymers, including polyethylene glycol (PEG) or polysaccharides, onto the PEI may help improve the stability of the polyplexes in serum (Pack et al., 2005; Laporte et al., 2006; Neu et al., 2001) and is one approach that

**3.2.5 pDNA/PEI polyplex transfection, DNA expression, and cell viability**

among coaxial electrospray, BM and LipofectamineTM 2000 (p<0.05).

The delivery efficiency of pGFP/PEI polyplexes produced by coaxial electrospray and bulk mixing was evaluated in NIH 3T3 cells and compared to LipofectamineTM 2000, the positive control. As illustrated in Figure 8(a), at N/P ratio of 6.7, cells transfected with polyplexes produced by coaxial electrospray showed similar GFP expression as those transfected with LipofectamineTM 2000, while cells transfected with polyplexes produced by bulk mixing showed much less GFP expression. No significant difference in cell viability was found

Since the evaluation of GFP expression is only qualitative, pSEAP was used to compare the delivery efficiency of polyplexes in a more quantitative way. As shown in Figure 9 (a), at N/P ratio of 6.7, cells transfected with the pSEAP/PEI polyplexes produced by coaxial electrospray gave 2.6 times higher SEAP expression than those produced by bulk mixing. Although cells transfected with LipofectamineTM 2000 showed the highest SEAP expression, 3.3 times higher than bulk mixing, the cell viability was low because of the toxicity of this

Both pGFP/PEI and pSEAP/PEI polyplexes were successfully produced using coaxial electrospray. The delivery efficiency of pDNA/PEI polyplexes was evalulated in NIH 3T3 cells. At N/P ratio of 6.7, polyplexes produced by coaxial electrospray were more effective at delivering genes to NIH 3T3 cells than those prepared by bulk mixing. Since coaxial

**3.2.4 Size distribution of pDNA/PEI polyplexes**

could reduce aggregation.

material. (Figure 9(b))

**3.2.6 Conclusions**

electrospray is an aerosol technique, it should be a useful way to deliver pDNA/PEI polyplexes directly to the lungs and achieve higher gene delivery efficiency than either electrospraying naked pDNA or nebulizing pDNA/PEI polyplexes produced by conventional methods.

Fig. 8. **(a)** The GFP expression in NIH 3T3 cells and **(b)** the cell viability measured 2 days post transfection. All polyplexes were produced at the N/P ratio of 6.7. For coaxial electrospray, the flow rate was 6mL/h and voltage was 5.0kV. (n=4)

Fig. 9. The pSEAP expression in NIH 3T3 **(a)** and the cell viability **(b)** measured 2 days post transfection. All polyplexes were produced at the N/P ratio of 6.7. For coaxial electrospray, the flow rate was 6 mL/h and voltage was 5.6 kV. (n=5, \*\*: p<0.005)

Electrospray Production of Nanoparticles for Drug/Nucleic Acid Delivery 235

The size distribution and surface charge of G3139 encapsulated lipoplexes prepared by coaxial electrospray depend on the concentrations and flow rates of G3139 solution and lipid mixtures. By adjusting operation parameters, the size of lipoplexes can vary between 100 nm and 2500 nm and the surface charge of lipoplexes can vary between -16 mV and +20 mV. (Wu et al. 2009b) The best balance between lipoplex productivity, size and surface charge, was achieved by setting the flowrates of the G3139 solution and the lipid mixture to 1.2 mL/h, the concentration of the G3139 solution to 0.5 mg/mL and the concentration of the lipid mixture to 10 mg/mL. Under these conditions Figure 10 shows that the lipoplex average mean diameter by volume was 190 ± 39 nm. The corresponding zeta potential was

<sup>100</sup> <sup>2</sup> <sup>3</sup> <sup>4</sup> <sup>5</sup> <sup>6</sup> <sup>7</sup> <sup>8</sup> <sup>9</sup>

Small angle neutron scattering (SANS) and cryo-transmission electron microscopy (cryo-TEM) were used to characterize the structures of G3139 encapsulated lipoplexes. Figure 11 illustrates typical SANS spectra for lipoplexes made by coaxial electrospray and by an

For the sample prepared by the ethanol dilution method, a Bragg Peak was observed, indicating the multi-lamellar structure of the lipoplexes, while the Bragg Peak was absent in lipoplexes produced by coaxial electrospray. From the position of the Bragg Peak we determined the inter-lamellar spacing *d* using the relation *d* = 2*q,* where *q* = ()sin(/2) is the momentum transfer vector, is the neutron wavelength, and is the scattering angle. When we fit the peak for the lipoplexes using a Gaussian function, we found a center-tocenter lamellar spacing of 6.6 +/- 0.2 nm. The cryo-TEM images support the SANS results by showing that most electrosprayed lipoplexes have a uni-lamellar structure, while lipoplexes prepared by ethanol dilution have onion like multi-lamellar structure with center to center inter-lamella distance measured from the picture of about 7-10 nm. The difference in the structure of lipoplexes produced by these two methods may reflect the removal rate of ethanol from the solution. In coaxial electrospray, the liquids break into tiny droplets, and

Fig. 10. A typical size distribution of G3139 encapsulated lipoplexes. The average mean

Dp, nm

 Lipoplex

1000

**3.3.3 Size distribution and zeta potential of G3139 encapsulated lipoplexes**

+4.5 ± 0.43 mV and the final lipid/G3139 ratio was 20.

100

80

60

Volume

diameter by volume was 190 ± 39 nm.

ethanol dilution method.

**3.3.4 Structure of G3139 encapsulated lipoplexes**

40

20

0

### **3.3 Coaxial electrospray lipoplexes for oligodeoxynucleotides (ODN) delivery**

#### **3.3.1 Introduction**

Lipoplexes, a major non-viral carrier system, have attracted a lot of research interest in the drug and nucleic acid delivery field. The phospholipid bilayer structure of lipoplexes provides great flexibility in encapsulating various drugs or nucleic acids, either within the hydrophobic bilayers or in the hydrophilic core. Many techniques, such as bulk mixing (Chrai et al. 2001; Elouahabi et al. 2005), the film method (Bangham method) (Fan et al. 2007; Otake et al. 2006) and alcohol dilution (Stano et al. 2004), have been developed to produce lipoplexes. However, non-uniform particle size, low encapsulation efficiency and multistep production process remain as major challenges. (Müller et al. 2000, 2004)

Antisense oligodeoxynucleotides (ODN) are short pieces of specially designed DNA. ODN has been widely investigated as a potential therapeutic agent against viral infections, cardiovascular inflammation, hematological malignancies, pulmonary diseases and cancer. (Chiu et al. 2006; Yang et al. 2004) ODN inhibits gene expression by hybridizing to specific mRNA sequences, which interferes with the target protein expression. For the preparation of ODN encapsulated lipoplexes, the ethanol dilution method is very popular (Stano et al. 2004; Jeffs et al. 2005). In this approach, the lipid mixture is first dissolved in ethanol and then mixed with the aqueous ODN solution. The final mixture is dialyzed against buffer solutions to remove ethanol and as the ethanol is removed ODN encapsulated lipoplexes are formed. The ethanol dilution method is clearly a batch production method, and batch to batch variation of the product can be an issue. In addition, multiple steps that may take days to complete are involved, and contamination of the final product can be an issue.