Tokai J Exp Clin Med., Vol. 31, No. 2, pp. 39-42, 2006

Efficient Preparation of Cationized Gelatin for Gene Transduction
Naoto FUKUYAMA, Tsuyoshi ONUMA＊ 1, Shio JUJO, Yoshifumi TAMAI ＊ 2, Takahiro SUZUKI ＊ 3,Kazunori MYOJIN＊ 4, Yasuhiko TABATA＊ 5, Yoshimi ISHIHARA＊ 1, Jiro TAKANO ＊ 1 and Hidezo MORI ＊ 6＊3

Department of Physiology, ＊ 2 Department of Radiation Oncology,
Department of Ophthalmology, ＊ 4 Department of Radiology, Tokai University School of Science＊1
Department of Chemistry, Tokai University School of Science＊5
Department of Frontier Medical Sciences, Kyoto University＊6
Department of Cardiac Physiology, National Cardiovascular Center(Received November 21, 2005; Accepted April 12, 2006)

We previously reported gene therapy using cationized gelatin microspheres of z 20-32 µm, prepared frompig skin, as a transducing agent, but although the gelatin offered various advantages, its yield was extremely
low (only 0.1%). In this study, we markedly improved the yield of z 20-32 µm cationized gelatin microspheres
and prepared a newly less than z 20 µm cationized gelatin. Conventionally, cationized gelatin is prepared bycationization, particulation by agitation, and cross-linking. The yield is determined by the particulation step,for which we had used a three-necked distillation flask of 500 mL and an agitation speed of 420 rpm. The
yield was significantly increased from 0.13 ± 0.02% to 8.80 ± 1.90% by using a smaller flask of 300 mL and anagitation speed of 25000 rpm (p ＜ 0.01). We could also prepare cationized gelatin of less than z 20 µm, which
had not been possible previously. We confirmed that efficient gene introduction into peritoneal macrophages
could be achieved with the new cationized gelatin.
Key words: gelatine microsphere, macrophage, yield

INTRODUCTION

MATERIALS

Efficient gene transduction methods are necessary
for gene therapy [1], and currently available methods
can be divided into viral vector techniques and nonviral approaches, such as lipofection or electroporation.
Viral vectors such as adenovirus or retrovirus offer
high transduction efficiency, but there are questions
regarding safety [2]. On the other hand, the efficiencyof transduction with non-viral vectors is generally poor
[3]. In recent years, nucleofection has been developed
for highly efficient gene transduction, but it can beapplied to only certain cells, and it causes damage insome cases [4].
We showed that intramuscular injection of FGF-4
gene-gelatin complex induced significantly greater
angiogenesis than injection of the bare FGF-4 gene[5]. Furthermore, we showed that adrenomedullin(AM) gene-gelatin complex effectively transduced theAM gene into endothelial progenitor cells (EPCs),
and the transduced EPCs had a therapeutic effect inpulmonary hypertension [6]. In those studies, we usedcationized gelatin microspheres of z 20-32 µm, derived
from pig skin. However, the yield of gelatin was only
about 0.1%. Cascone et al. reported that the preparationof nanoparticulate gelatin required an agitation speedof cationized gelatin and olive oil of more than 10,000rpm [7], so in this study, we examined whether the useof a higher agitation speed during preparation of thegelatin particles would increase the yield and reduce
the particle size in our procedure. We also confirmed
the efficacy of the gelatin particles thus obtained forgene transduction.

Gelatin of pig skin origin (PI 9) was purchasedfrom Nitta G elatin C orp, Japan. 1- Ethyl- 3 - (3 dimethylaminopropyl) carbodiimide (EDC), β-alanine,acetone, glycine, hydrochloric acid, ethylenediamine,olive oil, glutaraldehyde solution (GA), potassium
dihydrogenphosphate, disodium hydrogenphosphateand sodium hydrogen carbonate were purchased from
Wako Pure Chemical Industry, Japan. Liquid nitrogen
was purchased from Tomoe Corporation, Japan.
Rat peritoneal macrophages were collected by intraperitoneal injection of thioglycolate culture mediumas previously described [8]. DNA encoding GFP withthe cytomegalovirus enhancer-chicken β-actin hybrid
promoter was constructed [5].
METHODS
Conventional preparation of gelatin microspheres
involves three steps: (1) cationized gelatin production,(2) microsphere production and (3) cross-linking of
cationized gelatin microspheres. In order to increase theyield of cationized gelatin microspheres, we aimed toimprove the second step, i.e., microsphere production.
Cationized gelatin production
PI 9 (10 g) was completely dissolved in 0.1 M phosphate buffer (PBS, 450 mL) containing potassium dihydrogenphosphate and disodium hydrogenphosphate.
Ethylenediamine (31.1 mL) and hydrochloric acid wereadded, and the pH was adjusted to 5.0. EDC (5.35 g)
was added to this solution, which was made up to 500
mL with PBS and left for 18 hours. The solution was

Naoto FUKUYAMA or Hidezo MORI, Department of Physiology, Tokai University, Bohseidai, Isehara, Kanagawa 259-1193, Japan
Fax: +81-463-936-684 E-mail: fukuyama@is.icc.u-tokai.ac.jp― 39 ―

Tel: +81-463-931-121

N. FUKUYAMA et al. /Preparation of gelatin for gene transduction****

Fig. 1 Yield of z20-32 µm cationized gelatin using a 500
mL three-necked distillation flask.
The open bar shows the control (420 rpm) group,horizontal lined bar, the 570 rpm group and closedbar, the 25,000 rpm group. Data are presented asmean ± SEM. The yield in the 570 rpm group wassignificantly higher than that in the control group(*p ＜0.001 vs 420 rpm).

Fig. 2 Yield of z20-32 µm cationized gelatin using a 300
mL three-necked distillation flask.
The open bar shows the 420 rpm group, horizontal lined bar, the 570 rpm group and closedbar, the 25,000 rpm group. Data are presented asmean ± SEM. The yield in the 570 rpm group wassignificantly higher than that in the 420 rpm group(*p ＜ 0.001 vs 420 rpm). The yield in the 25,000rpm group was significantly higher than that in theother groups (**p ＜0.0001 vs other groups).

then dialyzed for 2 days with 16 changes of water.
After the dialysis, this solution was freeze-dried for 4 to
7 days to afford cationized gelatin. The conversion rateof carboxyl groups to amino groups was measured by
the TNBS method to characterize the product [13].

of cationized gelatin and 50 µg/100 µl gene (GFP orluciferase). The complex was incubated with rat peritoneal macrophages for 14 days. Effective gene introduction was demonstrated by cellular expression of GFP orluciferase.

Microsphere production
In the conventional procedure, cationized gelatinaqueous solution and olive oil were placed in a threenecked distillation flask of 500 mL at 40°C, andcentrifuged at 420 rpm for 10 minutes. This solution
was stirred for 30 minutes at 0°C. Acetone was added,and the mixture was centrifuged. After centrifugation,
the oil layer was removed, and acetone was added.
This solution was centrifuged again, homogenized andsieved with z 20 µm, z 32 µm and z 90 µm sieves (Testsieves, #JIS Z 8801, Tokyo Screen Co., Ltd., Japan).
The microspheres was dried in a refrigerator overnight,
and the yield of each fraction was determined.
In this experiment, we examined the effect ofincreasing the agitation speed to 570 rpm and 25000rpm, and the effect of using a smaller three-necked
distillation flask (300 mL) to obtain smoother mixing.
Cross-linking of cationized gelatin microspheres
Acetone and hydrochloric acid (7:3) were added
to cationized gelatin microspheres, the crosslinking
agent GA was added, and the reaction was allowed toproceed for 24 hours. After the reaction, centrifugation was performed and the supernatant was removed.
Glycine solution (100 mM) was added to remove GA
for one hour. After centrifugation, the supernatant wasremoved and cross-linked microspheres were cooledwith liquid nitrogen, freeze-dried, and weighed.
Gene introduction with the newly developed cationized gelatin microspheres
Gene gelatin complex was prepared by mixing 2 mg

RESULTS
Effect of agitation conditions on yield of cationizedgelatin
In the conventional method (500 mL/420 rpm), theyield of the z 20-32 µm cationized gelatin was 0.13 ±0.02%, but when the agitation speed was increased to570 rpm, the yield rose to 1.22 ± 0.52% (*p ＜ 0.001vs 420 rpm). The yield was further increased to 2.30
± 0.47% by increasing the agitation speed to 25000rpm from 570 rpm, but this further increase was notstatistically significant (p= 0.136)(Fig. 1). Next we useda smaller (300 mL) three-necked distillation flask withagitation at 420 rpm, 570 rpm and 25,000 rpm. Whenthe agitation speed was increased to 570 rpm, the yieldrose to 1.12 ± 0.49% from 0.11 ± 0.01% (*p ＜0.001vs 420 rpm). The yield was markedly increased to 8.80
± 1.90% by increasing the agitation speed to 25,000rpm from 570 rpm (**p ＜0.0001 vs 420 or 570 rpm)
(Fig. 2).
The yields of different-sized microsphere fractions
are summarized in Table 1. The use of the highest
agitation speed and the smaller flask allowed us to obtain cationized gelatin microspheres of less than z 20µm, which we had not been able to prepare with theconventional method, in addition to increasing the total
yield of the cationized gelatin microspheres.
Gene introduction with the new cationized gelatin
We examined the efficiency of the new, smallersized cationized microspheres for gene introduction
into rat peritoneal macrophages. As shown in Figure
3A, after coincubation of the macrophages and GFP― 40 ―

N. FUKUYAMA et al. /Preparation of gelatin for gene transduction

Table 1 Yields of different-sized cationized gelatines (%).

Flask size (mL) / Agitation speed (rpm)＞ z 20 µm

z 20-32 µm

500/4200500/570

0500/25000

0300/420

0300/570

0＊＊300/25000

2.12±0.21＊＊＊

Data are presented as mean ± SEM. p ＜0.001 vs 500/420,
p ＜0.0001 vs 300/420 or 300/570

＊＊＊

＊＊

z 90 µm ＞

0.13±0.02

15.1±3.21

14.0±5.30

1.22±0.52＊

13.2±3.52

14.1±6.13

2.30±0.47

17.2±4.10

7.23±1.60

0.11±0.01

19.1±5.32

17.4±7.12

1.12±0.49

22.1±7.62

11.3±2.52

8.80±1.90

32.2±11.0

9.10±2.10＊＊

z 32-90 µm＊＊＊＊＊p ＜0.001 vs 300/420*

Fig. 3 Gene introduction into rat peritoneal macrophages with cationized gelatin.
A. Upper column: Peritoneal macrophages were coincubated with GFP gene-cationized gelatin complex (M＋ GFP) for 14 days. GFP was expressed in the cytoplasm of the macrophages. Lower column:
Peritoneal macrophages were coincubated with cationized gelatin complex (M) for 14 days. GFP was not
expressed in the macrophages.
B. Luciferase activity. Peritoneal macrophages were coincubated with luciferase gene-cationized gelatincomplex (L ＋ G ＋ Mz, closed bar) or with cationized gelatin (G ＋ Mz, horizontal lined bar) or withno additive (Mz, open bar) for 14 days. Data are presented as mean ± SEM. The luciferase activity ofL+G+Mz was significantly higher than that of Mz or G+Mz ( ＊ p ＜0.01).

gene-cationized gelatin complex, the cells expressed
GFP. Coincubation of the macrophages and cationizedgelatin alone did not result in expression of GFP. As
shown in Figure 3B, after coincubation of the macrophages and luciferase gene-cationized gelatin complex,
the cells expressed luciferase activity of 1251 ± 257(counts/mg protein) on day 14, while the activity in thecontrol group, in which macrophages were cultured
alone, was only 5 ± 2 (counts/mg protein) (*p ＜0.01).
The luciferase activity was 8 ± 2 (count/mg protein)in the macrophage ＋ cationized gelatin group.

DISCUSSION
In this study, we showed that the yield of cationizedgelatin microspheres of z 20-32 µm increased with increasing agitation speed and with the use of a smaller
three-necked flask for the agitation of cationizedgelatin with olive oil. In addition, cationized gelatinmicrospheres smaller than z 20 µm could be prepared
for the first time with the highest agitation speed andthe smaller flask.
In the conventional method, 30% of cationized― 41 ―

N. FUKUYAMA et al. /Preparation of gelatin for gene transduction

gelatin finally formed microspheres, but almost all
were larger than z 32 µm [9]. Gene introduction withcationized gelatin microspheres involves cellular phagocytic activity, which is inefficient for particles as large
as z 30 µm [6]. By using a speed as high as 25,000rpm and a smaller flask (300 mL), we were able toincrease the yield of cationized gelatin microspheres ofz 20-32 µm to 8.80%. Furthermore, we could manufacture cationized gelatin microspheres smaller thanz 20 µm, which could not be obtained by the conventional method, in a yield of 2.12%.
Gedanken et al. succeeded in the production of
nanoparticles from various chemicals by using ultrasonic irradiation [10]. However, when we used anultrasonic homogenizer for agitating cationized gelatin
and olive oil, the yield decreased to 1.30% .
We previously showed that our gelatin microspheregene complexes were introduced into cells by phagocytosis. Since the efficiency of cellular phagocytotic
activity is greater for smaller particles, the developmentof smaller-sized cationized gelatin microspheres isexpected to increase the efficiency of gene introduction
via phagocytosis. Further, Kaul et al. showed that gene
introduction into fibroblasts, which exhibit endocytosis
but not phagocytosis, was possible by using nanoparticles of polyethylene glycol [11]. Therefore, if ourmethod can be extended to obtain cationized gelatinmicrospheres in the nano size range, the variety of cells
to which they would be applicable may be considerably
extended. It is still the case that a gene introduction
method with adequate safety and efficiency for clinicalapplication is not yet available [12]. The ingredient,gelatin, used in this study is already in clinical use,
and is considered to be safe. Cationized gelatin microspheres cannot be used to introduce genes into alltypes of cells, and the efficiency of gene introduction
is lower than that of viral vectors. However, the use ofcationized gelatin microspheres to introduce a gene into
endothelial progenitor cells did have an apparent andprolonged therapeutic effect [5, 6]. The smaller cationized gelatin microspheres developed in this study may
provide increased efficiency of gene introduction into
various cells.
In conclusion, we have improved the preparation of
cationized gelatin microspheres for gene transduction,
obtaining a greater yield, as well as smaller microspheres, which may have clinical potential.
ACKNOWLEDGEMENT
This work was supported by grants from Tokai
University School of Medicine Research Aid in 2004and 2005, the research and study program of Tokai
University Educational System General Research

Organization and Kanagawa Nanbyou foundationin 2004, as well as a Grant-in-Aid for Scientific
Research in 2003 (No. 15659285) and 2005 (No.
17659375) from the Ministry of Education, Scienceand Culture, Japan and Health and Labour Sciences
Research Grants for Research on Human Genome,
Tissue Engineering Food Biotechnology in 2003 (H15saisei- 003). Health and Labour Sciences Research
Grants for comprehensive Research on Cardiovascular
Diseases in 2004 (H16-jyunkannki(seishuu)-009).
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