Statistical analysis

Results were expressed as mean ± SE. Statistical significance of differences between mean values was estimated using the Student’s t-test (Microsoft Excel).

p < 0.05 was considered significant.

3. Results

3.1 Physical characterization of Liposome-polymer transfection complex The liposome-polymer transfection complex (LTPC) was constructed from two hydrophilic polymers (PEG, PEI) and soybean oil. After sonication, LPTC could be rapidly produced and appeared as white emulsion (Fig. 2).

Sequentially, the sizes of LPTC were measured by dynamic light scattering (DLS) and the results showed that the major particle sizes were distributed between 212.2 nm and 320.1nm. And the second range was from 1099.5 nm to 1658.9 nm (Fig. 3). The surface charge of the particles showed cationic property and the strength was +38.6 mv measured by zeta-sizer. Besides, the complex’s pH was measured and the value was 10.98 (data not shown).

The structure of LPTC was also observed by transmission electron microscopy (TEM), which showed hollow structure and round shape (Fig. 4a).

Furthermore, the outer surface of the particles were high dense as hair-like structure (Fig. 4b). The hair-like structure was considered as made by polymers.

3.2 The effect of DNA bound to LPTC on particle size and zeta-potential The cationic LPTC may bind to anionic DNA via electrostatic interaction.

Thus, different amounts of LPTC were reacted with 50 mg DNA to observe the changes on their particle size and zeta-potential. As the amounts of LPTC were

increased, the sizes of LPTC/DNA complexes were firstly increased from 1 to 5 of N/P ratio, in contrast to the higher N/P ratio (10 and 30) that would decrease their sizes (Fig. 5a). In addition to the changes of zeta-potentials (surface charge), the surface charges were firstly decreased from 1 to 5 of N/P ratio and then increased at 10 to 30 of N/P ratio (Fig. 5b). We also captured the appearance of LPTC/DNA complex by TEM (Fig. 6).

3.3 Gel retardation of LPTC/DNA complex

The binding capability of DNA is essential for gene transferring. Hence, the DNA binding ability of LPTC was examined by agarose gel electrophoresis in different N/P ratios (Fig. 7a). The result showed that at N/P ratios higher than 5, there was no band on the gel. It referred that plasmid DNA was adsorbed by LPTC. Moreover, heparin was also used to compete with the electrostatic interaction of DNA and LPTC (Fig. 7b). However, when the complexes were incubated with higher dosage of heparin, the migration of DNA was observed obviously at N/P ratios ranging from 1 to 30. Therefore, the results indicated that DNA did bind to LPTC.

3.4 Protective effect of LPTC/DNA complex on DNase I digestion

To investigate whether DNA complexed to LPTC could be protected from DNase I digestion was further proceeding. Hence, LPTC/DNA complex was treated with DNase I and examined the integrity of DNA bound on LPTC (Fig.

8). Gel electrophoresis revealed that DNA alone without LPTC adsorption was completely degraded by DNase I but not affected by heparin treatment (lanes 7 and 8). In the counter part, LPTC/DNA complexes treated with DNase I (lane 2) showed the same intensity band as untreated LPTC/DNA complex (lane 1). In addition, heparin was added to release bound DNA from LPTC with or without DNase I treatment (lane 3 and 5), then these results showed that DNA bound on LPTC was not affected by DNase I. By contrast to the bound DNA released from LPTC by heparin was digested by DNase I (lane 4). Therefore, we proved that LPTC showed the ability to protect bound DNA from nuclease’s digestion.

3.5 In vitro transfection efficiency of LPTC/DNA complex

In the in vitro transfection tests, LPTCs were complexed with plasmid pAAV-MCS-hrGFP DNA encoded green fluorescent protein as reporter gene and then transfected into Balb/3T3 cells in the absence of serum conditions. By different N/P ratios, the reporter gene expressions were low efficiency (2% or 13% at 1 or 5 N/P ratio), in contrast to high trasnfection efficiency 32% or 63%

at 10 or 30 N/P ratio (Fig. 10). However, the gene expressions of green fluorescent protein were observed by fluorescent microscopy and the results showed at N/P ratio of 5 and 10 had high expression (Fig. 11). The results indicated that N/P ratio of LPTC could affect the transfection efficiency and the expression of transfectants.

Furthermore, the cytotoxicity of LPTC in Balb/3T3 cells was also measured by MTT assay and the results showed that the dosage of LPTC at N/P ratio of 30 and 10, cell viability was 70.4% and 85%, respectively (Fig. 9).

3.6 Cellular uptake of LPTC/BSA-FITC complex

Here, whether LPTC could enhance cellular uptake was investigated. BSA conjugated FITC was complexed with different dosage of LPTC to interact with macrophage cells (P338/D1). The intracellular fluorescence represented the degree of internalization. At 37°C, the cellular uptake efficiency of LPTC/BSA-FITC complex was increased 38.7% compared with BSA-LPTC/BSA-FITC alone (Fig. 12a).

However, cellular uptake efficiency of LPTC/BSA-FITC complex was not different with BSA-FITC alone at 4°C (Fig. 12b).

3.7 LPTC induces TNF-alpha secretion in splenocytes of naïve mice

Naïve mice splenocyte were taken out and treated with LPTC for 48 h, then TNF-alpha secretion were detected in supernatant by cytokine ELISA. The result showed that LPTC could stimulate TNF-alpha secretion in splenocytes and show higher concentration (105 pg/ml) of TNF-alpha secretion than control group (26 pg/ml) (Fig. 13).

3.8 The in vivo adjuvant effects of LPTC on heterologous immunization

In vitro studies showed that LPTC could enhance the gene transferring efficacy, the cellular uptake of antigen by macrophage cells and also stimulate the pro-inflammatory cytokine (TNF-alpha) secretion. Thus, we further investigated whether LPTC could be an effective vector to deliver DNA or antigen in vivo. Firstly, a heterologous immunization protocol was developed by priming with DNA and boosting with protein antigen to investigate the effect of LPTC on the induction of humoral response (Fig. 15). Specific anti-Hphsp60 responses and anti-Urease B responses were measured, respectively. The results showed that the treatment of LPTC complexed with DNA or antigen could enhance about 1-fold of anti-Hphsp60 Ig responses compared to immunization without LPTC facilitation (Fig.16a). Furthermore, LPTC complexed with 25 µg of DNA could increase anti-Hphsp60 Ig responses and show to enhance about 2-fold of Ig responses compared to 12.5 µg of DNA at weeks 4 (Fig.16b). By contrast, the anti-Urease B Ig responses were both induced at low levels at 2nd and 4th weeks (Fig. 17a, 17b). Thus, we continued to booste of 100 µg DNA with or without LPTC formulation (Fig. 18). Both of LPTC formulated groups enhance about 1 –fold of anti-Urease B Ig response.

3.9 The in vivo adjuvant effect of LPTC on co-delivery immunization

However, what a vaccine strategy could induce antigen specific immune response in the short time was an important issue in clinical trials. Here, co-delivery immunization protocol was developed by prime or boost with DNA and

protein antigen at the same time to investigate the effect of LPTC on the induction of humoral response (Fig. 19). After 2 weeks of last boost, the results showed that the treatment of LPTC complexed with DNA and protein antigen could enhance about 2-fold of anti-Hphsp60 Ig responses (Fig. 20) and about 3-fold of anti-Urease B Ig responses compared to immunization without LPTC facilitation (Fig. 21).

4. Discussion

In recent years, the development of vaccine was mostly focused on liposome that can encapsulate the antigens within the lipid bilayer. The encapsulated antigens would slowly release from the vesicle to continuously stimulate host immunity, which can trigger more efficient immune responses than non-capsulated antigens. In addition, Korsholm KS and colleagues also demonstrated that cationic liposome-capsulated antigen will facilitate the antigen uptake and presentation [17]. Furthermore, cationic liposomes have more potency than anionic or neutral liposomes in inducing cell-mediated immune response to soluble proteins [16]. Besides, the various phospholipids had differential adjuvant activities to induce protective immunity. Together these results, they suggested that the structures or components of liposomes may be determined the strengths of the immune responses or adjuvant effects.

Liposome-based vaccines have been investigated in human trials including vaccines against malaria, HIV, hepatitis A and influenza and these vaccines were safe and highly immunogenic [20]. However, liposome-based vaccines for animal are few and far between in vaccine development. In the intensive farm of livestock breeding, the prevention of infectious disease would become very important to avoid economic loss. Thus, how to develop an inexpensive and an efficient liposome-based animal vaccine would be an important issue to apply in intensive farm of livestock [12]. Here, we developed a novel liposome-polymer

transfection complex (LPTC) which have the components including high molecular weight branched polyethylenimine (PEI, 25 kDa) (Fig. 1), polyethyleneglycol (PEG) and soybean oil. The cationic polymer, PEI was an effective transfection reagent and had some particular advantages such as, (1) the high cationic charge-density potential is able to compact DNA efficiently for enhancement of DNA delivery [10], and (2) its “proton-sponge” effect could release DNA from the endosome to protect transgenes from degradation [11].

The other hydrophilic molecules polymer, polyethylenglycol (PEG) has been used to coat liposome and the PEG coating liposome could significantly diminish the uptake by macrophage of liver and spleen leading to an increase blood circulation time [21]. Generally, the third component is soybean oil that composed of three unsaturated fatty acid (linolenic acid, linoleic acid and oleic acid) and two saturated fatty acid (stearic acid and palmitic acid). Here, soybean oil was used to form liposome structure via sonication and may also provide an modification that is suitable for extra addition of immunostimulatory components such as the saponin adjuvant Quil A, which is derived from the bark of the South American Quillaia saponaria Molina tree and has been used as component of immunostimulating complex (ISCOM) [22]. The immunostimulatory agents typically showed the hydrophobic nature and immunogenic property, hence easily mixing with soybean oil through apolar interactions to improve the adjuvant effect of vaccine. In addition, all of these components in LPTC are very inexpensive, which can largely reduce the cost of

animal vaccine and suit to apply in massive inoculation of animal vaccine for prevention of infectious disease between animals.

However, for gene delivery vectors included cationic liposome, cationic polymer and polycationic liposome, they should be examined in the following criteria: (1) DNA adsorptive ability, (2) the efficiency of gene delivery, (3) low cytotoxic, and (4) large-scale commercial manufacture. These criteria were important for efficient gene expression and possible practical application. Here, we constructed the novel LPTC with the satisfactions for above criteria and offered an efficient and rapid preparation procedural to produce the gene delivery vectors. We used LPTC that showed high positive charge to absorb DNA and formed smaller complexes. In addition, the complexes showed the higher efficiency of gene transferring in N/P ratio of 10 and 30. Besides, we found LPTC showed the adjuvant effect and efficiently enhanced antigen specific antibody responses in both strategies of immunization (heterlogous and codelivery).

The structure and dimensions of LPTC were round, nanometer scale for size (Fig. 4) and showed the positive charge. We speculated the positive charge that was arisen from the cationic polymer (PEI) and it may exist on the surface of particles as the high density of hair-like filaments (Fig. 4b). The assumption of the interaction between PEI polymers and liposome was due to the hydrophobic force between large branched chains of PEI and lipid layer. Furthermore, the dimensional changes and surface charge changes occurring by varying the N/P

ratios of LPTC/DNA complex formation was examined (Fig. 5). Here, the result showed higher N/P ratio that could form smaller complexes and we presumed that DNA rigidly bound on LPTC at the existence of large amounts of LPTC.

And also another view pointed that high positive charge may reduce the aggregation by electronic repulsion, which helpt the smaller LPTC/DNA complexes formation. Furthermore, for positive charged complex in surface charge detection, which can facilitate adherence to cellular membranes, inducing and increasing intracellular uptake.

A successful gene therapy or DNA vaccine relies on an efficient DNA liberation from the endocytotic vesicles and the following DNA nuclear localization for final gene expression. However, D Lechardeur1 et al. proposeed that cytosol nucleases are responsible for the rapid degradation of plasmid DNA, which limits the ability of DNA nuclear localization [23]. Our data shown that LPTC was not only complexed with DNA efficiently in high N/P ratio but also protected DNA from the DNase I digestion (Fig. 7 and Fig. 8). Godbey et al have been implied that the protection of DNA by PEI resulted from a physical or electrostatic barrier to enzymatic degradation with DNAse I [24]. Thus, we speculated that PEI exhibited an important role in LPTC for DNA complexation and the protection of DNase I degradation.

Furthermore, in vitro transfection results showed that as the N/P ratios increasing could increase the transfection efficiency (Fig. 9). The gene expression could be easily visualized at N/P ratio of 5 and 10. (Fig. 10c,d).

However, the higher transfection efficiency was at N/P=30 but exhibited low gene expression in fluorescence microcopy observation (Fig. 10e). The possible explanation was that less DNA bound on LPTC in large amounts of LPTC existence, so less DNA entered into each cell. Nevertheless, large amounts of LPTC enhanced the probability of LPTC/DNA complexes entering into cells.

We considered N/P ratio of 10 that showed the highest gene expression in this result, it was the most suitable charge ratio of LPTC/DNA complex for following DNA vaccine treatment in vivo studies.

Cationic lipid and cationic liposome had been used as vaccine adjuvants included DNA vaccines and protein-based vaccines [25, 26]. Thus, antigen adsorbed on cationic LPTC was investigated whether could effectively enhance the cellular uptake of antigen-presenting cells (APCs). We showed that LPTC could enhance the efficiency of cellular uptake (Fig. 11a). We presumed that the cationic property of LPTC was good for targeting to the cell membrane of antigen-presenting cells, which subsequently leads to enhanced uptake. The explanation was also examined and confirmed the primary adjuvant effect of cationic liposome by other studies [17].

A variety of DNA vaccine prime and recombinant viral boost immunization strategies have been developed to enhance immune responses in humans. This prime and boost vaccination strategy has been used to overcome the ineffective induction of immune responses displayed by DNA immunization alone in nonhuman primates and humans [21]. And furthermore, the advantages of this

approach include a synergistic effect on the induction of immune responses and the generation of a robust T cell-mediated immune response [27]. Here, we used LPTC as a dual vector to carry DNA for priming immunization and carry protein for boosting immunization, and indeed enhanced higher antigen specific immune response. However, the animal vaccine strategy should be considered that how to induce higher immune response in short time for wide range of animals. Hence, we explored the possibility of efficiently co-delivering DNA vaccine and protein-based vaccine by carrying with LPTC. And the result showed that this vaccine strategy was successful to stimulate immune response after once immunization and induced higher immune response of twice immunization (Fig. 18). Both of the immune response after once and twice immunization in co-delivery vaccine strategy showed higher antibody response than heterologous immunization strategy. Furthermore, the co-delivery vaccine strategy was also confirmed by other studies and was examined to prime an enhanced and balanced specific immunity of Th1 and Th2-biased responses [28].

Thus, theses results confirm the usefulness of LPTC in in vitro and in vivo gene delivery, and provide the adjuvant effect. Furthermore, LPTC showed the potential to apply in the development of animal vaccine for farm animals that need large quantity of vaccine product with low cost to prevent the infectious disease, particularly for species where a large number of animals with a relatively low commercial value are utilized such as chickens.

Figures

Figure 1. Structure of branched polyethylenimine (bPEI)

The branched form of PEI shows a theoretical ratio of primary (1°) to secondary (2°) to tertiary (3°) nitrogen atoms of 1:2:1. Every third atom of PEI is a nitrogen atom capable of protonation. [10]

1°

2°

3°

1° 1°

STEP 3: LPTC formation STEP 1: Polymers solution

Soybean oil

Storage in 4°C refrigerator STEP 2: Two immiscible phases

PEI and PEG aqueous solution

Before

Sonication

After

Figure 2. The liposome-polymer transfection complex (LPTC) preparation STEP 1: Polymers solution, both of PEI and PEG were well dissolved in ddH₂O to form aqueous phase. STEP 2: Two immiscible phases, soybean oil was added to the polymer solution that formed two separate phases. STEP 3:

LPTC formation, the solution was sonicated for 30 min that formed a uniform phase and showed the milky white appearance. PEI: polyethylenimin; PEG:

polyethylene glycol.

Figure 3. The particle distribution of LPTC

The particles distribution was measured by dynamic light scattering (DLS). Y axis was represented the relative numbers of particles and X axis was represented diameter (nm) of particles. The particle size was distributed into two populations, the major population was from 212.2 nm to 320.1nm and the minor range was from 1099.5 nm to 1658.9 nm.

Figure 4. Transmission electron microcopy of LPTC particles

In Fig. 4(a), LPTC particles showed a round shape and particle size were ranging from 100 nm to 300 nm. In high magnification (Fig. 4b), the arrow pointed the hair-like filaments that existed on the outer surface of LPTC.

(a) (b)

Figure 5. Particle size and zeta-potential of LPTC/DNA complexes

In Fig. 5a and 5b, different dosages of LPTC were mixed with 50 µg DNA and then formed different N/P ratios in order of 1 to 30. After DNA incubated with LPTC for 30 min, the particle size (Fig. 5a) and zeta-potential (Fig. 5b) was measured by Zeta-sizer. NC represented the LPTC alone.

(a) (a) (b)

Figure 6. Transmission electron microcopy of LPTC/DNA complex

LPTC/DNA complex photograph was took by TEM and the black arrow pointed the DNA fragment bound on LPTC. The size of complex was about 1.5µm.

Figure 7. Gel retardation assay of LPTC/DNA complexes

In Fig. 7a, LPTC at different concentrations were incubated with a fixed amount of DNA for 30 min at room temperature, and the complexes were ran on an agarose gel. Lane 1 was DNA marker and lane 2 was naked DNA, lane 3 to lane 7 were represented at N/P ratios from 1, 2, 5, 10 and 30, respectively. The displacement of DNA from complexes by heparin competition was shown on Fig. 7b. DNA complexes after treatment were analyzed with 0.8% agarose gel electrophoresis.

Fig. 3. Agarose gel electrophoresis retardation assay of OIA/DNA binary complexes with increasing of N/P ratios with or without heparin.

In Fig. 3 (a), the plasmid DNA is condensed compactly at N/P ratios higher than 5 without heparin. However, in Fig. 3 (b), when the complexes were incubated with heparin, the migration of DNA is observed obviously at N/P ratios ranging from 5 to 30. It was shown that the interaction between OIA and DNA is via electrostatic interaction.

Fig. 3. Agarose gel electrophoresis retardation assay of OIA/DNA binary complexes with increasing of N/P ratios with or without heparin.

In Fig. 3 (a), the plasmid DNA is condensed compactly at N/P ratios higher than 5 without heparin. However, in Fig. 3 (b), when the complexes were incubated with heparin, the migration of DNA is observed obviously at N/P ratios ranging from 5 to 30. It was shown that the interaction between OIA and DNA is via electrostatic interaction.

1 2 3 4 5 6 7 1 2 3 4 5 6 7

(a) (b)

Figure 8. LPTC protects DNA from DNase I digestion.

LPTC protected DNA from DNase I digestion was assessed by treatment with DNase I, DNase I and heparin, DNase I then heparin and heparin alone.

LPTC/DNA complexes were formed at N/P=10 with 300ng of DNA. DNA complexes after treatment were analyzed with 0.8% agarose gel electrophoresis.

1.Untreated 2.DNase 1 3.DNase 1 + Heparin 4.Heparin + DNase 1 5.Heparin 6.Untreated 7.DNase I 8.Heparin

LPTC/DNA complexes DNA

Figure 9. Cytotoxicity tests of LPTC on Balb/3T3 cells

Cell viability of cells treated with different dosages of LPTC were measured by MTT assay. Cells were incubated with various dosages of LPTC and cell viability was measured at 72 hr after treatment. Data represent the percentage to untreated cell.

Figure 10. In vitro Transfection efficiency of LPTC/DNA complexes

Using 3 µg DNA and different dosages of LPTC formed each N/P ratio in order from 1 to 30. LPTC/DNA complexes transfected into Balb/3T3 cells, after 48 hr the transfection efficiency was measured by flow cytometry. NC: cell alone, Data represents the mean ± S.E. of six experiments. **P<0.01 v.s.

Negative Control

**

**

Figure 11. Fluorescent microscopy of gene expression after in vitro transfection

Cell were transfected with each LPTC/DNA complex in order of 1 to 30.

Forty-eight hours after transfection, phase contrast image (A), fluorescent microscopy images (B) of transfectants were monitored (200x magnification).

Forty-eight hours after transfection, phase contrast image (A), fluorescent microscopy images (B) of transfectants were monitored (200x magnification).