New Methods for the Characterization of Different Gelatin Based Nanoparticulate Drug Carrier Systems

J. Zillies, G. Winter, C. Coester

Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-University Munich, Butenandtstraße 5, D-81377 Munich, Germany

Introduction Results and Discussion

Due to their promising properties in drug delivery and drug To establish a simple and safe fractionation technique, preliminary 1,20
leading to a baseline separation of the oligonucleotide from the void
targeting, highly biocompatible nanoparticulate carrier experiments were performed applying the Capillary Hydrodynamic peak (peak 3). A sufficient separation could also be achieved
1,00
systems made of gelatin are of major interest. In contrast to Fractionation (CHDF). The obtained results show the method´s when applying a lower initial cross-flow level (peak 4). The
great advances made in the formulation and possible general applicability but without providing a sufficient nanoparticle / displayed peak sizes represent different analyte

UV-Signal (volt)
0,80

applications, adequate characterization of the developed oligonucleotide baseline separation (Fig. 3). In addition, a direct concentrations. In addition to the influence of the
0,60
systems remains a challenging aspect. In addition to the correlation between length and inner diameter of the applied concentration a higher cross-flow causes generally a decreasing
description of the dimensions of (un-) modified nanoparticles capillaries and the fractionation results was demonstrated [3]. 0,40 detection signal.
this also includes monitoring the course and extent of drug 0,20 According to the experiments accomplished with the ssDNA
loading reactions. Drugs are often loaded to nanoparticulate oligonucleotide, the data obtained combining the siRNA
4,00 0,00
drug carrier systems by ionic or hydrophilic / hydrophobic 0 5 10 15 20 25 30 35 40 oligonucleotide and gelatin nanoparticles to achieve a drug loading
3,50 gelatin nanoparticles
interactions. This presentation reveals the binding capacity of Elution Time (min)
reaction are contradictory to our expectations (Fig. 6). We found a
3,00
positively charged gelatin nanoparticles for a ssDNA UV-Signal (volt) slight decrease in the blank test oligonucleotide signal after
2,50 Fig. 4 Fractionation of ssDNA oligonucleotide (____ 100 µg/ml) and gelatin
oligonucleotide and a small interfering dsRNA (siRNA) saturating the ultrafiltration membrane with gelatin nanoparticles,
oligonucleotide nanoparticles (____ 500 µg/ml) before and after (____) drug loading; initial
oligonucleotide. 2,00
cross-flow 65%, cross-flow duration 780 sec (cp. Fig. 2 ____), 1 kDa cut-off which can also be verified performing drug loading trials.
1,50
The aim of this study is to analyze the mentioned drug ultrafiltration membrane Furthermore, after drug loading, there is a clear decrease of the
1,00
loading reaction based on ionic interactions by means of nanoparticles´ signal instead of an increase. These results indicate
0,50
Asymmetrical Flow Field-Flow Fractionation (AF4). In this The results obtained for the blank test of the double stranded siRNA some interactions between siRNA and nanoparticles, as well as,
0,00
context, the AF4-Technology offers some unique features 0 10 20 30 40 50 60 oligonucleotide are shown in Fig. 5. with the ultrafiltration membrane and membrane saturating
which approves it for this particular application. The Elution Time (min)
0,30
nanoparticles, respectively, based upon the analytes´ ionic
determination of small molecular weight analytes next to properties.
dispersions up to the micrometer area is possible, as well as, Fig. 3 Fractionation of ssDNA oligonucleotide (10µg/ml) and gelatin 0,25

nanoparticles (476µg/ml); capillary set-up: length 20m, inner diameter 4

sampling directly from the reaction tube. The underlying

UV-Signal (volt)
0,30
0,20
0,13 mm, flow-rate 0.05 ml/min
fractionation mechanism is shown in Fig. 1. 0,25
0,15
1
Similar studies to those using CHDF were performed with AF4.

UV-Signal (volt)
Injection Fractionation Elution 0,20
0,10
Optimal conditions for nanoparticle / oligonucleotide fractionation
0,15
were developed using model proteins with a MW similar to those of 0,05
2 3
the siRNA and DNA oligonucleotides, respectively (data not shown). 0,00
0,10

With the combination ssDNA oligonucleotide and gelatin 0 5 10 15 20 25 30 35 40

0,05
Elution Time (min)
nanoparticles, a positive drug loading reaction could be
characterized. Fig. 5 siRNA oligonucleotide blank test, initial cross-flow 65% 75% (____),
0,00
0 5 10 15 20 25 30 35 40

The results of the drug loading process are shown in Fig. 4. The (____) and 60% (____), cross-flow duration 780 sec (cp. Fig. 2 ____/ ____/ ____) Elution Time (min)

Cross-Flow level of oligonucleotide load on the cholamin modified nanoparticles

Fig. 1 Fractionation mechanism of AF4
To achieve a sufficient separation in order to determine the
Fig. 6 Fractionation of siRNA oligonucleotide (20,45 µg/ml) and gelatin
can be determined by interpretation of the according UV-signals. Peak 1 displays the adaptation of the fractionation conditions as nanoparticles (448,8 µg/ml), initial cross-flow 60%, cross-flow duration 780
The difference in AUC of loaded nanoparticles and AUC of unloaded described above to the siRNA oligonucleotide. With an increased sec (cp. Fig. 2 ____), 5 kDa cut-off ultrafiltration membrane
nanoparticles corresponds to the differences of the oligonucleotide initial cross-flow intensity the separation from the void peak, inherent ____ oligonucleotide before drug loading and before nanoparticulate saturation

drug loading capacity of the gelatin nanoparticles, it is AUCs. The required linear correlation between the AUC of the to AF4 systems, will be enhanced (peak 2). The higher MW of the of the ultrafiltration membrane
necessary to establish fractionation conditions that provide a separated analytes and their varying concentration was verified siRNA oligonucleotide compared to the ssDNA oligonucleotide ____ oligonucleotide before drug loading and after nanoparticulate saturation

baseline separation of gelatin nanoparticles and the
oligonucleotides, allowing a quantitative assessment of the
measured AUC values obtained for each single analyte.
Another point to be met for the quantitative assessment is the
linearity between concentration of the sample solutions and
resulting AUCs of the detection signals.
References
[1] Coester, C., von Briesen, H., Langer, K., Kreuter, J., Gelatin
experimentally (data not shown). permit the application of a 5 kDa cut-off ultrafiltration membrane of the ultrafiltration membrane
____ gelatin nanoparticles before drug loading
____ oligonucleotide and gelatin nanoparticles after drug loading
Materials and Methods
A preliminary saturation of the ultrafiltration membrane or the use of
a more hydrophobic membrane material are considered as the next
The study was performed using a HRFFF-10.000 AF4 system sequence) was kindly provided by Prof. A.M. Vollmar, Department attempts to overcome the described drawbacks.
(Postnova Analytics, Landsberg a. L., Germany), consisting of a of Pharmacy, Center of Drug Research, Pharmaceutical
separation channel, autosampling system, in-line degasser and on- Biology, Ludwig-
line solvent filter (0.1 µm, PTFE). The channel height was set at 500 Maximilians- 80
Conclusion
µm. The regenerated cellulose ultrafiltration membranes had a cut- University Munich, 70

nanoparticles by two step desolvation - a new preparation method, off of 1 kDa and 5 kDa, resp. (Nadir Filtration, Wiesbaden, Germany. The 60
C ro ss F lo w (% )

surface modifications and cell uptake, (2000) J. Microencapsulation, 17,

Germany). Detection was performed using UV-spectrophotometry applied 50 Our results showed that AF4 is a promising new analytical method
187-194 for the separation of gelatin nanoparticles from low molecular weight
[2] Coester, C., Development of a new carrier system for (wavelength 260 nm, Spectra System UV 1000, Thermo Separation fractionation 40

Products, Germany). PBS buffer pH 7.4 was used as eluent. Gelatin conditions specified 30 payloads. The nanoparticle loading with single stranded DNA
oligonucleotides and plasmids based on gelatin nanoparticles, (2003)
New Drugs, 1, 14-17 nanoparticles (250 nm) were prepared by a two-step desolvation by cross-flow
20
oligonucleotides could be quantified. Further investigations have to
[3] Zillies, J., Coester C., Capillary Hydrodynamic Fractionation (CHDF)
method [1] and partly surface modified with cholamin as described intensity and
10
be carried out concerning the difficulties reported for the
0
and other semi-chromatographic methods as new analytical tools for the previously [2]. The DNA oligonucleotide (18-mer, single stranded, duration are shown 0 300 600 900 1200 1500 1800 2100 2400 nanoparticle loading with double stranded siRNA oligonucleotides.
separation and analysis of protein based nanoparticles aside low Time (sec)
Moreover, by coupling AF4 with adequate detection systems like
molecular weight proteins and oligonucleotides, (2003) Proceedings of
MW 5451 Da) was purchased from MWG Biotech GmbH, in Fig. 2. The
Ebersberg, Germany. The siRNA oligonucleotide (21-mer, double channel net-flow Fig. 2 AF4 fractionation conditiones multi-angle light scattering an overall characterization of
the CRS Local Chapter Annual Meeting, April 4, Ludwig-Maximilians-
University Munich, Germany stranded, MW 6710,2 Da sense sequence and 6590,2 antisense was set at 1ml/min. nanoparticles is easily possible.

2003 AAPS Annual Meeting and Exposition, October 26th – 30th, Salt Lake City, Utah, USA