Plasmid DNA Encapsulation Using an Improved ... - ACS Publications

Chapter 16

Plasmid DNA Encapsulation Using an Improved Double Emulsion Process Edmund J . Niedzinski*, Yadong Liu, and Eric Y. Sheu

Downloaded by CORNELL UNIV on July 7, 2012 | http://pubs.acs.org Publication Date: February 16, 2006 | doi: 10.1021/bk-2006-0923.ch016

Genteric, Inc., 50 Woodside Plaza, Suite 102, Redwood City, CA 94061 *Corresponding author: [email protected]

The route of administration for an active pharmaceutical ingre dient (API) often requires the development of novel formu lations to facilitate effective delivery. Many API, for example most proteins, polypeptides, oligonucleotides and plasmid DNA, degrade under extreme pH or in the presence of digestive enzymes. In order to successfully deliver these API orally, formulations that are able to protect and deliver them to the targeted sites are mandated. One common strategy for protecting API is encapsulation, using biocompatible poly meric materials. The encapsulation materials and the methods used determine the stability of the formulation and the bio availability of the API. We have developed a novel method for the encapsulation of plasmid DNA into a compatible poly mer. This method produces particles that are smaller than those made by conventional methods, encapsulate plasmid DNA with greater than 90% efficiency, protect plasmid DNA from degradation, and have release characteristics similar to convential nonviral delivery methods. These cumulative data suggest that this delivery system may be suitable for delivery of DNA into harsh environments.

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© 2006 American Chemical Society

In Polymeric Drug Delivery I; Svenson, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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Introduction The effective delivery of an active pharmaceutical ingredient (API) requires a thorough understanding of the properties of the API and the obstacles that the API encounters during its application. Since many API are vulnerable to chemi cal and biological obstacles, advanced formulations are required for effective delivery. For example, conventional oral delivery is not possible for most proteins, polypeptides, oligonucleotides and plasmid DNA due to the low pH in the stomach and enzymatic degradation along the enteric route. In order to orally adminster sensitive API, formulations that protect and deliver them to the targeted sites are mandated. The encapsulation of an API into a biocompatible polymeric material, such as poly(lactide-co-glycolide), is a common strategy to protect and deliver sensitive API. As the field of gene therapy grows, novel formulations for plasmid DNA are needed (1,2). A successful gene therapy product will require a delivery system that can remain stable and successfully deliver a genetic payload to the target cells. Although a number of nonviral gene therapy formulations are in clinical trials (3), many of these formulations may not tolerate harsh conditions. There fore, a more robust system would extend the applicability of gene therapy. To meet this goal, we have developed a polymeric encapsulation strategy that would withstand the harsh chemical and biological obstacles that are encountered in many common routes of delivery.

Figure 1. Overview of Double Emulsion Formulation Process. The double emulsion process shown in Figure 1 is a convenient method to generate particles for drug delivery (4). While this method was originally de signed to encapsulate water-soluble polypeptides and small molecule drugs (5), this method can also be used to encapsulated DNA. Unfortunately, the use of double emulsions to encapsulate hydrophilic bio-therapeutics such as proteins and plasmid DNA results in relatively low encapsulation efficiency and high particle size polydispersity (6). Although this particle size range may be suitable


230 for delivery to antigen presenting cells, the uptake of particles by these cells limits the clinical application of this technology. Through the incorporation of cationic lipids, we have modified the double emulsion process to produce submicron particles with greater than 90% enapsulation efficiency. Herein, we present the development of this technology.

Materials and Methods


Materials 50:50 poly(D,L-lactic-c0-glycolic) acid (PLGA) was purchased from Birmingham Polymers. Zwittergent 3-14 was purchased from Aldrich. Poly vinyl alcohol) was purchased from Sigma (30-70kDa), Aldrich (13-23 kDa), Fluka (31 kDa) and Alfa Aesar (22-26 kDa). DNA was prepared as an endotoxin-free reduced, supercoiled plasmid using anion exchange resins (Qiagen) at Genteric, Inc. Cationic lipids were purchased from Avanti Polar Lipids (Birmingham, AL). Representative Formulation Procedure An aqueous DNA solution (0.5 mg of plasmid DNA in 0.75 mL TE buffer) is added to a solution of polymer (50 mg of 50:50 poly(lactic-co-glycolic) acid) in CH C1 (1 mL) to form a water-in-oil (w/o) emulsion. This mixture is emulsified by vortexing at 2500 rpm for 15 sec. A proper quantity of A B M is added and the emulsion is further mixed by vortexing (2500 rpm/15 sec). The resulting emulsion is added to an aqueous solution (8% PVA, 25 mL) to form a water-in-oil-in-water (w/o/w) emulsion. The solution is allowed to stir (1500 rpm) until the CH C1 evaporates, resulting in a solid particle. The particles are collected by centrifuging (4000 rpm, 15 min.). The supernatant is decanted and the particles are washed with water (25 mL). This process is repeated three times and the microparticles are lyophilized. The particles are then collected and stored at 0°C. The supernatant and wash solutions are collected to be analyzed for DNA concentration. 2

2

2

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Particle Size The particle size was determined by examining the microparticles under 400x magnification on a Micromaster optical microscope (Fisher Scientific), equipped with a Kodak MDS290 documentation system.


231 Encapsulation Efficiency The relative encapsulation efficiency of the microparticles was assessed by measuring the amount of DNA in the supernatant and washes of the microparticle preparation. This solution was diluted with 1% Zwittergent 3-14 in TAE buffer and then analyzed using the PicoGreen® reagent (Molecular Probes). The assay was conducted according to the manufacturer's instructions, using a Gemini XS Fluorescence Microplate Reader (Molecular Devices).


Preparation and Purification of Reporter Genes The plasmid pBATSEAP contains the secreted alkaline phosphatase (SEAP), operably linked to human cytomegalovirus major immediate early enhancer/promoter, which is positioned upstream of the first intron of human (3globin. pBATLuc is an analogous plasmid, in which the SEAP gene was replaced with the luciferase gene. The plasmid vectors were produced by bacterial fermentation and purified with an anion exchange resin (Qiagen, Santa Clarita, CA) to yield an endotoxin-reduced, supercoiled plasmid, containing less than 100 E.U./mg DNA as measured by clot L A L assay (Charles River Endosafe). Stock DNA solutions were prepared using sterile water. Analysis ofEncapsulated Plasmid DNA A microparticle sample (2-5 mg) in a 4-mL glass vial was treated with CH C1 (1 mL) and gently mixed for 16 hours. The suspension was treated with a solution of 1% Zwittergent in TAE Buffer (pH 7.4) and the resultant emulsion was gently mixed for 4 hours. The sample was allowed to stand until the two phases separate, and the aqueous layer was tested for DNA concentration using the PicoGreen® reagent (Molecular Probes). The assay was conducted accor ding to the manufacturer's instructions, using a Gemini XS Fluorescence Microplate Reader (Molecular Devices). 2

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Cell culture CHO cells from the American Type Culture Collection (ATCC, Rockville, MD) were cultured in 75 cm cell culture flasks with HAMS F12 media con taining 10% bovine calf serum. NIH3T3 cells were grown in Dulbecco's modi fied Eagle's medium with 10% fetal calf serum. The cells were maintained at 37°C in a 5% C 0 environment. Cells were split into 24-well plates at 60% con fluence 24 hours before transfection. 2

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Transfection of Cultured Cells Growth media was removed from the CHO cells and replaced with 100|iL of Dulbecco's modified Eagle's medium with or without 10% fetal calf serum. The


232 formulations were diluted with water to obtain a DNA concentration of lmg/ lOOmL and 100|iL was administered to each well. Liposome formulations were prepared as previously described. After 2 hours, the formulation solution was removed, frozen for DNA analysis, and replaced with 500jLiL of Dulbecco's modified Eagle's medium with or without 10% fetal calf serum. After 24 hours, the medium was removed,frozenfor SEAP analysis, and replaced with 500|nL of fresh growth medium. This process was repeated at 48 hours and 120 hours.


Secreted Alkaline Phosphatase Assay Tissue supernatants, prepared in luciferase lysis buffer, were analyzed for SEAP concentration using the chemiluminescent SEAP Reporter Gene Assay available from Roche Diagnostics (Indianapolis, IN). Briefly, the samples were diluted 1:50 with Dilution Buffer then incubated in a water bath for 30 min at 65°C. The samples were then centrifuged for 1 min at 13,000 rpm and 50 \\L of the resulting supernatant was transferred to a microtiter plate. 50 | i L of the provided Inactivation Buffer was added, followed by a 5-minute incubation at room temperature. The Substrate Reagent (50 |LiL) was added prior to a 10minute incubation at room temperature. Light emissions from the samples were measured using an L-max plate luminometer from Molecular Devices (Sunnyvale, CA). The relative light units were converted to mass of SEAP pro tein, based on a standard curve run contemporaneously with the samples. Plas ma concentrations of SEAP were measured using the same assay, except that the samples were diluted 1:7 with the Dilution Buffer.

Results and Discussion The addition of a cationic lipid (DSTAP) into a double emulsion signifi cantly increases the efficiency of plasmid DNA encapsulation (Figure 2). The efficiency increases as more cationic lipid is added to the double emulsion, with a three-fold increase for a formulation containing DSTAP:DNA in a 4:1 ratio compared to a formulation without DSTAP. Besides increasing the encapsulation efficiency, the addition of a cationic lipid to a double emulsion has a profound effect on the size of PLGA micro particles. As shown in Figure 3, increasing the DSTAP.DNA ratio resulted in a population of particles that were smaller and less polydisperse. The micro particles with DSTAP were approximately 1-3 urn in size, while the particles without DSTAP were noticeably larger (5-10 um), as determined by light microscopy. However, increasing the charge ratio beyond 4:1 did not result in an increase in encapsulation efficiency or decrease in particle size (data not shown).


233 100%

Encapsulation Efficiency


0 % -I 0

. 1

. 1 2 3 Charge Ratio (DSTAP:DNA)

1 4

Figure 2. Effect of cationic lipid concentration on DNA encapsulation efficiency. Particles were generated using different concentrations of DSTAP. The amount of DNA contained in the microparticles was measured using Pico-Green®. The encapsulation efficiency was calculated as a percentage of DNA found in the supernatant samples relative to amounts of DNA that was added to the formulation.

Figure 3. Effect of cationic lipid concentration on particle size. Particles were generated using different concentrations of DSTAP, and particle size was analyzed using light microscopy.


234 The encapsulation efficiency of this formulation appeared to be influenced by the cationic lipid structure (Table I). Specifically, adding cationic lipids with the same cationic head group and longer hydrophobic domains slightly increase the encapsulation efficiency. Interestingly, the inclusion of cationic lipids with different structures had no observable effect on the particle size (data not shown). Table I. Effect of Cationic Lipid Structure on DNA Encapsulation Efficiency.


3

Cationic Lipid None DMTAP (C14:0) DPTAP (CI 6:0) DSTAP (C18:0)

Encapsulation Efficiency 30.38% 90.38% 91.92% 92.69%

a

The encapsulation efficiency was calculated as a percentage of the DNA found in the supernatant samples relative to amounts of DNA that was added to the formulation. The particles were formulated at 4:1 charge ratio. Although the double emulsion process is conducted under relatively mild conditions, high stir rates and temperatures can degrade sensitive API. To deter mine if any decomposition occurred during formulation, the integrity of the encapsulated plasmid DNA was analyzed using agarose gel electrophoresis. As shown in Figure 4, plasmid DNA that was extracted from the microparticles appears to be slightly nicked in comparison to control plasmid, a phenomona that has been observed in other studies (7). Fortunately, subsequent studies revealed that the encapsulated plasmid retained full biological activity in cell culture. To test the ability of these formulations to protect DNA under degradative conditions, the formulations were challenged by incubating with DNase type I (Figure 5). Control plasmid DNA and formulated plasmid DNA was incubated for 20 minutes in buffer and various concentrations of DNase type I. After incubation, the plasmid DNA was extracted from the particles and the structural integrity of the plasmid DNA was analyzed using gel electrophoresis. After incubating for 20 minutes with 1.6 units/mL of DNase I, the encapsulated plasmid remained intact while the control plasmids was completely degraded.



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Figure 4. Agarose gel electrophoretic analysis ofplasmid DNA. To remove the polymeric coating, three identical microparticle samples (1, 2, and 3) were incubated with CH Cl for 16 hours. A 1% solution of zwittergent in TE buffer was used to extract the plasmid DNA and disrupt any plasmid DNA cationic lipid association. An aliquot of this aqueous layer was then analyzed using a 0.8% agarose gel Samples were concurrently analyzed with control plasmid. 2

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Figure 5. Analysis of DNA stability following incubation with DNase type I. Particle formulated with DSTAP at 4:1 charge ratio were incubated with various concentrations ofDNase Ifor 20 minutes at 40 °C. Control samples were incubated in TAE buffer. The DNA was extractedfrom the particles and analyzed using gel electrophoresis.

The gene transfer efficiency of the particles was assessed by treating cultured Chinese Hamster Ovary (CHO) cells with particles containing secreted alkaline phosphates (SEAP). Encapsulated plasmid DNA and control con ditions were administered to CHO cells in 24-well tissue culture plates. First, DNA uptake was analyzed by measuring the amount of DNA that remained in the supernatant 2 hours after administration (Figure 6). The highest concen tration of plasmid DNA was found in the supernatant of cells that were treated with DNA in water. Significantly less DNA was detected in the other solutions (8). After 24 hours of incubation, the supernatant was collected and measured for SEAP expression (Figure 7). This process was repeated at 48 and 72 hours after administion. Microscopic analysis of the cell population did not reveal any observable toxicity. Interestingly, the levels of SEAP expression from cell treated with DSTAP liposomes are similar to the cells treated with the micro particle formulation.


237 1.6


Water

DNA

Lipid

Particles

Formulation

Figure 6. Analysis of extracellular DNA concentration. Formulations were tested by administering 100 pL to each well containing CHO cells in 100 juL of serum positive (+) media. After 2hours, the media/formulation was removed and frozen for analysis. The samples were then thawed and testedfor DNA concentration using Pico-Green® (samples diluted 1/10 in 1% zwittergent in TAE buffer). Each data point represents the average of three wells +/- SD. Assay sensitivity

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