U.S. patent application number 10/513429 was filed with the patent office on 2005-08-04 for non-viral gene delivery system.
This patent application is currently assigned to FMC Biopolymer AS. Invention is credited to Artursson, Per, Christensen, Bjorn Erik, Koping-Hoggard, Magnus, Varum, Kjell Morten.
Application Number | 20050170355 10/513429 |
Document ID | / |
Family ID | 19913595 |
Filed Date | 2005-08-04 |
United States Patent
Application |
20050170355 |
Kind Code |
A1 |
Artursson, Per ; et
al. |
August 4, 2005 |
Non-viral gene delivery system
Abstract
The present invention concerns a composition comprising
complexes of cationic chitosan oligomers derived from the cationic
polysaccharide chitosan, wherein said cationic oligomers contain a
weight fraction of less than 20% of oligomers with a Degree of
Polymerization (DP)<10 in addition to a weight fraction of less
than 20% with DP>50, and a nucleic acid. These compositions
comprising well-defined cationic chitosan oligomers having a
certain distribution of chain lengths, and nucleic acid are
advantageous to achieve delivery of the nucleic acid into cells of
a selected tissue, and to obtain in vivo expression of the desired
molecules encoded for by the nucleic acid.
Inventors: |
Artursson, Per; (Uppsala,
SE) ; Christensen, Bjorn Erik; (Trondheim, NO)
; Koping-Hoggard, Magnus; (Uppsala, SE) ; Varum,
Kjell Morten; (Trondheim, NO) |
Correspondence
Address: |
CONNOLLY BOVE LODGE & HUTZ, LLP
P O BOX 2207
WILMINGTON
DE
19899
US
|
Assignee: |
FMC Biopolymer AS
P.O. Box 494- Brakeroya
Drammen
NO
N-3002
|
Family ID: |
19913595 |
Appl. No.: |
10/513429 |
Filed: |
November 3, 2004 |
PCT Filed: |
May 2, 2003 |
PCT NO: |
PCT/NO03/00143 |
Current U.S.
Class: |
435/6.13 ;
536/20; 536/23.1 |
Current CPC
Class: |
A61P 37/00 20180101;
C12N 15/87 20130101; A61P 37/06 20180101; A61K 9/0073 20130101;
A61P 31/00 20180101; A61P 35/00 20180101; A61K 48/0041 20130101;
A61K 47/61 20170801 |
Class at
Publication: |
435/006 ;
536/020; 536/023.1 |
International
Class: |
C12Q 001/68; C08B
037/08; C07H 021/02 |
Foreign Application Data
Date |
Code |
Application Number |
May 3, 2002 |
NO |
2002 2148 |
Claims
1. A composition comprising complexes of: (a) cationic chitosan
oligomers derived from the cationic polysaccharide chitosan wherein
said cationic oligomers contain a weight fraction of less than 20%
of oligomers with a Degree of Polymerization (DP)<10 in addition
to a weight fraction of less than 20% with DP>50; and (b) a
nucleic acid.
2. The composition of claim 1, wherein said cationic chitosan
oligomers are obtained from chitosan using chemical or enzymatic
methods.
3. The composition of claim 1, wherein said cationic oligomers
contain preferably a weight fraction of less than 20% of oligomers
with DP<12 in addition to a weight fraction of less than 20%
with a DP>40 and most preferably a weight fraction of less than
20% of oligomers with DP<15 in addition to a weight fraction of
less than 20% with a DP>30.
4. The composition of claim 1, where the fraction of N-acetylated
units (F.sub.A) of said chitosan oligomers is less than 0.60,
preferably less than 0.35, more preferably less than 0.1 and most
preferably less than 0.01.
5. The composition of claim 1, wherein said composition essentially
has a net positive charge ratio.
6. The composition of claim 1 wherein said chitosan oligomers are
derivatized with targeting ligands and stabilizing agents.
7. The composition of claim 1, wherein said complexes comprise a
coding sequence that will express its function when said nucleic
acid is introduced into a host cell.
8. The composition of claim 7, wherein said nucleic acid is
selected from the group consisting of DNA and RNA molecules.
9. The composition of claim 8, wherein said composition has a pH in
the range of 3.5 to 8.
10. The composition of claim 9, wherein said composition after
aerosolisation essentially has a comparable droplet size as to a
composition consisting of only nucleic acid at equal concentrations
of nucleic acid.
11. A method of preparing the composition of claim 1, comprising
the steps of: (a) exposing said cationic chitosan oligomer to an
aqueous solvent; (b) mixing the aqueous solution of step (a) with
said nucleic acid in an aqueous solvent; and (c) reducing the
volume of the product solution obtained in step (b) to achieve a
desired concentration of the said composition.
12. A method of administering nucleic acid to a mammal, using the
composition of claim 1, and introducing the composition into the
mammal.
13. The method of claim 12, wherein the composition is introduced
into the mammal by administration to mucosal tissues by pulmonary,
nasal, oral, buccal, sublingual, rectal or vaginal routes.
14. The method of claim 12, wherein the composition is introduced
into the mammal by administration to submucosal tissues by
parenteral routes that is intravenous, intramuscular, intradermal,
subcutaneous or intracardiac administration, or to internal organs,
blood vessels or other body surfaces or cavities exposed during
surgery.
15. A method of claim 12 comprising the composition of claim 1,
whereby said nucleic acid is capable of expressing its function
inside said cell.
16. The use of the composition of claim 1, in the manufacture of a
medicament for prophylactic or therapeutic treatment of a mammal,
or in the manufacture of a diagnostic agent for use in in vivo or
in vitro diagnostic methods.
17. The use of the composition of claim 16 in the manufacture of a
medicament for use in gene therapy, antisense theraphy, or genetic
vaccination for prophylactic or therapeutic treatment of
malignancies, autoimmune diseases, inherited disorders, pathogenic
infections and other pathological diseases.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a new non-viral delivery
system for nucleic acids, and more specifically, to a system, which
facilitates the introduction of nucleic acid into cells in a host
tissue after administration to that tissue. This system is based on
a composition comprising chemically and physical-chemically
well-defined cationic chitosan oligomers derived from biodegradable
chitosan polysaccharides that efficiently delivers biologically
active nucleic acids, such as oligo or polynucleotides that encodes
a desired product, and facilitates the expression of a desired
product in cells present in that tissue.
BACKGROUND ART
[0002] The concept of gene therapy is based on that nucleic acids,
DNA, RNA can be used as pharmaceutical products to cause in vivo
production of therapeutic proteins at appropriate sites. Delivery
systems for nucleic acids are often classified as viral and
non-viral delivery systems. Because of their highly evolved and
specialized components, viral systems are currently the most
effective means of DNA delivery, achieving high efficiencies for
both delivery and expression. However, there are safety concerns
for viral delivery systems. The toxicity, immunogenicity,
restricted targeting to specific cell types, limited DNA carrying
capacity, production and packaging problems, recombination and a
very high production cost hamper their clinical use (Luo and
Saltzman, 2000). For these reasons, non-viral delivery systems have
become increasingly desirable in both basic research laboratories
and clinical settings. However, from a pharmaceutical point of
view, the way of delivery of nucleic acids still remains a
challenge since a relatively low expression is obtained in vivo
with non-viral delivery systems as compared to viral delivery
systems (Saeki et al., 1997).
[0003] A variety of non-viral delivery systems, including cationic
lipids, peptides or polymers in complex with plasmid DNA (pDNA),
have been described in the prior art (Boussif et al., 1995; Felgner
et al., 1994; Hudde et al., 1999). The negatively charged nucleic
acids interacts with the cationic molecules mainly through ion-ion
interactions, and undergo a transition from a free form to a
compacted state. In this state the cationic molecules may provide
protection against nuclease degradation and may also give the
nucleic acid-cationic molecule complex surface properties that
favour their interaction with and uptake by the cells (Ledley,
1996).
[0004] Among these cationic molecules, the synthetic polymer
polyethylenimine (PEI) have been shown to form stable complexes
with pDNA and mediate relatively high expression of the transgene
both in vitro and in vivo (Boussif et al., 1995; Ferrari et al.,
1997; Gautam et al., 2001). For this reason, PEI is often used as a
reference system in the experimental setup. However, a rough
correlation between toxicity and efficiency has been suggested for
PEI (Luo and Saltzman, 2000) and recent studies have addressed
concerns about toxicity using PEI (Godbey et al., 2001 Putnam et
al., 2001). Another drawback with PEI is that it is not
biodegradable and it may therefore be stored in the body for a long
time. Therefore, the search for effective and non-toxic
biodegradable non-viral delivery systems is highly desirable.
[0005] Most commonly, non-viral delivery systems have been
delivered in vivo by the parenteral route. After intravenous
administration to mice, compacted nucleic acid-cationic molecule
complexes deposited mainly in the lung capillaries where the gene
was expressed in the endothelium of the capillaries in the alveolar
septi (Li and Huang, 1997; Li et al., 2000; Song et al., 1997) or
even in the alveolar cells (Bragonzi et al., 2000; Griesenbach et
al., 1998), but not in the epithelium. However, unformulated, naked
DNA was rapidly degraded in the blood circulation before it reached
its target and generally resulted in no gene expression. In
contrast, injection of naked DNA into skeletal muscle resulted in a
dose-dependent gene expression (Wolff et al., 1990) which was
further enhanced when complexed with a non-compacting but
`interactive` polymer such as polyvinyl pyrrolidone (PVP) or
polyvinyl alcohol (PVA) (WO 9621470) (Mumper et al., 1996; Mumper
et al., 1998). Thus, gene transfection in vivo is tissue-dependent
in an unpredictable way and therefore remains a challenge.
[0006] Mucosal delivery of non-viral delivery systems has also been
described that is delivery to the gastrointestinal tract, nose and
respiratory tract (Koping-Hoggard et al., 2001; Roy et al., 1999),
WO 01/41810. With exception for the delivery to the nasal tissue
where DNA in uncompacted form gives the best gene expression (WO
01/41810) compacted nucleic acid-cationic molecule complexes are
preferred to uncompacted DNA when a high gene expression is
required in a mucosal tissue.
[0007] In prior art, non-viral gene delivery systems are based on
cationic polymers such as chitosan of rather high molecular weight,
often several hundred kilodaltons (kDa) with 5 kDa as a lower
limit, see for example MacLaughlin et al., 1998, Roy et al., 1999
and WO 97/42975. The major reason is that polymers of lower
molecular weight (<5 kDa) form unstable complexes with DNA,
resulting in a low gene expression (Koping-Hoggard, 2001). However,
there are many drawbacks using cations of high molecular weight
such as increased aggregation of compacted nucleic acid-cationic
molecule complexes and solubility problems (MacLaughlin et al.,
1998). Further, there are several biological advantages of using
cationic molecules of lower molecular weights that is they
generally show reduced toxicity and reduced complement activation
compared to cations of higher molecular weights (Fischer et al.,
1999; Plank et al., 1999).
[0008] In the prior art some examples of the use of low molecular
weight cations for complexation with nucleic acid have been
described (Florea 2001; Godbey et al., 1999; Koping-Hoggard, 2001;
MacLaughlin, et al., 1998; Sato et al., 2001). However, these low
molecular weight cations form unstable compacts with DNA that
separate in an electric field (agarose gel electrophoresis)
resulting in no or a very low gene expression in vitro, as compared
to cations of higher molecular weights. This can be explained by
that complexes formed between DNA and low molecular weight cations
are generally unstable and dissociate easily (Koping-Hoggard,
2001). In fact, the dissociation of cationic molecule-DNA compacts
and release of naked DNA during agarose gel electrophoresis has
often been used as an assay to distinguish ineffective formulations
from effective ones in the literature (Fischer et al., 1999;
Gebhart and Kabanov, 2001; Koping-Hoggard et al., 2001). Then, it
is known from the prior art that complexes between DNA and cations
should be stable to mediate a high gene expression.
[0009] The prior art contains various examples of methods for the
delivery of nucleic acids to the respiratory tract using non-viral
vectors (Deshpande et al., 1998; Ferrari et al., 1997; Gautam et
al., 2000). We recently identified and characterized one such
system based on the DNA-complexing polymer chitosan (Koping-Hoggard
et al., 2001), a linear polysaccharide, which can be derived from
chitin. Chitosan-based gene delivery systems are also described in
U.S. Pat. No. 5,972,707 (Roy et al., 1999), WO 98/01160 and in US
Patent Application no. 2001/0031497 (Rolland et al., 2001).
[0010] Chitosan has been introduced as a tight junction-modifying
agent for improved drug delivery across epithelial barriers
(Artursson et al., 1994). It is considered to be non-toxic after
oral administration to humans and has been approved as a food
additive and also incorporated into a wound-healing product (Illum,
1998).
[0011] Chitosans comprise a family of water-soluble, linear
polysaccharides consisting of (1.fwdarw.4)-linked
2-acetamido-2-deoxy-.be- ta.-D-glucose (GlcNAc, A-unit) and
2-amino-2-deoxy-.beta.-D-glucose, (GlcN, D-unit) in varying
composition and sequence (FIG. 1). The definition adopted here to
distinguish between chitin and chitosan is based on the
insolubility of chitin in dilute acid solution and the solubility
of chitosan in the same dilute acid solution (Roberts, 1992).
[0012] The relative content of A- and D-units may be expressed as
the fraction of A-units:
F.sub.A=number of A-units/(number of A-units+number of D-units)
[0013] F.sub.A is related to the percentage of de-N-acetylated
units through the relation:
% de-N-acetylated units=100%.multidot.(1-F.sub.A)
[0014] Each D-unit contains a hydrophilic and protonizable amino
group, whereas each A-unit contains a hydrophobic acetyl group. The
relative amounts of the two monomers (that is
A/D=F.sub.A/(1-F.sub.A)) can be varied over a wide range, and
results in a broad variability in their chemical, physical and
biological properties. This includes the properties of the
chitosans in solution, in the gel state and in the solid state, as
well as their interactions with other molecules, cells and other
biological and non-biological matter.
[0015] The influence of the chemical structure of chitosans was
recently demonstrated when chitosans were used in a non-viral gene
delivery system (Koping-Hoggard et al., 2001). Chitosans of
different chemical compositions displayed a structure dependent
efficiency as gene delivery system. Only chitosans that formed
stable complexes with pDNA gave a significant transgene expression.
Such complexes required that at least 65% of the chitosan monomers
were deacetylated.
[0016] Chitosans can be depolymerized either chemically or
enzymatically to obtain chitosan polymers or oligomers of the
desired molecular size. Various chemical degradation mechanisms can
be used to depolymerize chitosans, that is acid hydrolysis, nitrous
acid and oxidative-reductive depolymerization. Ultrasonic
depolymerisation of polymers may alternatively be used, but these
methods are very inconvenient for producing very low molecular
weights. Depolymerisation of chitosan by the use of nitrous acid is
a convenient way of preparing low-molecular weight chitosan, as
described in for example U.S. Pat. No. 3,922,260 and U.S. Pat. No.
5,312,908. This mechanism involves deamination of a D-unit, forming
2,5-anhydro-D-mannose unit at the new reducing end, which can be
reduced to 2,5-anhydro-D-mannitol using NaBH.sub.4 as shown in FIG.
2. Alternatively, various enzymes can also be used to depolymerize
chitosan, for instance U.S. Pat. No. 5,482,843, chitosanases,
chitinases, and lysozyme. Also acid hydrolysis may be used to
depolymerise chitosan (V.ang.rum et al., 2001, and references
therein).
[0017] In the prior art, studies of the effect of molecular weight
of chitosan on transfection efficiency in vitro of chitosan-pDNA
complexes showed no significant dependence of the molecular weight
in the size range 20-200 kDa (Koping-Hoggard et al., 2001;
MacLaughlin et al, 1998). However, in another study (Sato et al.,
2001) chitosans of 15 kDa and 52 kDa showed higher gene expression
than chitosan >100 kDa, while no gene expression was detected
with a 1.3 kDa chitosan. Further, studies of gene expression in
vitro and in lung tissue in vivo using a series of low molecular
weight chitosans (1.2 kDa, 2.4 kDa and 4.7 kDa) showed that only
the 4.7 kDa chitosan mediated a significant gene expression
(Koping-Hoggard, 2001).
[0018] Chitosans of different molecular weights have been used as
components in complexes for non-viral gene delivery. For example,
US patent application no. 2001/0031497A refers to the use of small
molecular weight chitosan as a component of the delivery system,
that is chitosan in the range of 24 kDa Mw, which resulted in the
smallest particle of gene delivery system and also in an increased
transfection of cells with the condensed delivery system in
vitro.
[0019] Chitosans of different molecular weights which are used in
gene delivery systems are normally unfractionated samples obtained
from commercial suppliers, and lower molecular weights are obtained
from said samples by partial degradation using degradation agents
such as organic or inorganic acids, nitric acid or chitosan
degrading enzymes. In all cases, the distribution of molecular
weights remains relatively high. As an example, a commercial
chitosan with a weight average molecular weight (M.sub.w) of
180.000 was analysed by size-exclusion chromatography using a
refractive index detector and a multi-angle laser light scattering
detector. FIG. 3A shows the elution profile, that is refractive
index detector signal, which is proportional to the concentration
of chitosan, combined with a plot of the calculated molecular
weight (expressed as chitosan in the acetate salt form) as a
function of the elution volume. It is evident that the sample
contains molecular weights as high as 10.sup.6 g/mol (1000 kDa) at
the beginning of the peak and as low as 14 (10 kDa) at the end of
the peak. A recalculation of these data gives the cumulative
molecular weight distribution (FIG. 3B). It may be inferred from
these calculations that 12% (w/w) of the sample has a molecular
weight below 40 kDa and 38% of the sample has a molecular weight
below 100 kDa. Likewise, 18% of the sample has a molecular weight
above 300 kDa and 9% has a molecular weight above 400 kDa. The
sample is thus polydisperse since it contains polymers of different
molecular weights or chain lenghts.
[0020] Chitosans may be supplied in the free amine form or as
different salts such as chitosan chloride, chitosan glutamate and
chitosan acetate. The salt-form influences the relationship between
the molecular weight (M) and DP (the number of sugar residues per
molecule). The following equations describe this relationship
between DP and M:
1 Free base: M = DP (161(1 - F.sub.A) + 203F.sub.A) = DP (161 +
42F.sub.A) Chitosan M = DP (197.45(1 - F.sub.A) + 203F.sub.A) = DP
(197.45 + 5.55F.sub.A) chloride: Chitosan M = DP (221(1 - F.sub.A)
+ 203F.sub.A) = DP (221 - 18F.sub.A) acetate: Chitosan M = DP
(308(1 - F.sub.A) + 203F.sub.A) = DP (308 - 105F.sub.A)
glutamate:
[0021] The weight average molecular weight (M.sub.w) of a
polydisperse sample may be expressed as
M.sub.w=.SIGMA.c.sub.iM.sub.i/.SIGMA.c.sub.i where c.sub.i is the
concentration (g/l) of a particular molecular weight (M.sub.i)
within the distribution) (Tanford, C. (1961) Physical chemistry of
macromolecules, John Wiley and Sons, New York, Section 8b).
Likewise, the number average molecular weight (M.sub.n) may be
expressed as M.sub.n=.SIGMA.c.sub.i/.SIGMA.(c.sub.i/M.sub.i). In
the case referred to above M.sub.w=180 kDa and M.sub.n=84.5 kDa,
and the polydispersity index which is defined as M.sub.w/M.sub.n
equals 2.1. A polydispersity near 2 is characteristic of a linear
polymer which has been subjected to random depolymerisation
(Tanford, C. (1961) Physical chemistry of macromolecules, John
Wiley and Sons, New York, Section 33a).
[0022] The distribution of chain lenghts following a random
depolymerisation of a linear polymer such as chitosan is given by
the equation (Tanford (1961):
W.sub.x=xp.sup.x-1(1-p).sup.2
[0023] W.sub.x is the weight fraction of chains containing x
monomers (for chitosan the monomers are sugar residues) and p is
the fraction of intact linkages and 1-p is the fraction of cleaved
lingages. The number average degree of polymerisation (x.sub.n)
equals 1/(1-p). Since M.sub.n=M.sub.0x.sub.n, where M.sub.0 is the
monomer equivalent weight, which is 203 g/mol for a residue of
N-acetyl-glucosamine when it occurs within a chitosan chain and 161
g/mol for a residue of glucosamine in the free base form when it
occurs within a chitosan chain. For a given F.sub.A the average
M.sub.0 becomes equal to 203.multidot.F.sub.A+161.mul-
tidot.(1-F.sub.A).
[0024] FIG. 4 shows SEC-MALLS chromatograms (4A), and differential
(4B) and cumulative (4C) molecular weight distributions of a
chitosan, which has been depolymerised by nitrous acid to obtain
different weight average molecular weights in the range from 41.500
to 13.400. It is clearly shown that the calculated molecular weight
distributions remain broad. These data clearly demonstrate that
chitosans of different molecular weights which are produced from a
high molecular weight by partial degradation remain polydiserse and
contain chains of widely differing molecular weights.
[0025] The molecular weight distribution of a polymer may be
modified by selectively removing certain parts of the distribution.
Chitosan samples with relatively short chains may be fractionated
by gel filtration to obtain individual oligomers or fractions with
relatively narrow molecular weight distributions. One example is
given by T.o slashed.mmeraas et al. (2001) who obtained purified
chitosan oligomers in the range of 2-10 residues per chain.
[0026] Samples with higher molecular weights may also be
fractionated by gel filtration as demonstrated for chitosans by
Ott.o slashed.y et al. (1996). Typically, fractions with
M.sub.w/M.sub.n values of 1.2-1.5 was obtained by fractionating a
normally polydisperse sample with M.sub.w=270.000 using a gel
filtration column containing Sepharose CL-4B and Sepharose
CL-6B.
[0027] In an alternative method polydisperse chitosans may be
fractionated by dialysis or membrane techniques which allow
selective removal of the shortest chains, and where the resulting
distribution depends on the initial distribution as well as the
membrane characteristics porosity and transport coefficients and
the operating conditions.
[0028] According to the present invention it was surprisingly
discovered that chitosans of a single chain lenght or chitosans
with narrow molecular weight distributions had different properties
as complexing agents in gene delivery than other samples of
comparable M.sub.w or M.sub.n, but with broader molecular weight
distributions.
[0029] Another disadvantage of many cations used for complexation
of nucleic acid e.g. PEI, polylysine and chitosan is that they are
roughly processed bulk chemicals with a broad molecular weight
distribution and hence rather undefined (Godbey et al., 1999). It
is well established that such chemicals may display a batch to
batch variation. Therefore, from a pharmaceutical point of view,
well-defined polycations having a narrow molecular weight
distribution are preferred.
[0030] Another disadvantage using broad molecular weight
polycations for complexation of nucleic acids and subsequent
transfection is that chains of differents lenghts may have
different complexation and transfection effectivities.
SUMMARY OF THE INVENTION
[0031] The present invention is concerned with a composition
comprising complexes of:
[0032] (a) cationic chitosan oligomers derived from the cationic
polysaccharide chitosan wherein said cationic oligomers contain a
weight fraction of less than 20% of oligomers with a Degree of
Polymerization (DP)<10 in addition to a weight fraction of less
than 20% with DP>50; and
[0033] (b) a nucleic acid.
[0034] According to the present invention it has unexpectedly been
found that compositions comprising well-defined cationic chitosan
oligomers having a certain distribution of chain lengths, and
nucleic acid are advantageous to achieve delivery of the nucleic
acid into cells of a selected tissue and to obtain in vivo
expression of the desired molecules encoded for by the nucleic
acid.
[0035] It is another object of the invention to provide a method of
preparing compositions according to the invention, comprising the
steps of
[0036] (a) exposing said cationic chitosan oligomers to an aqueous
solvent,
[0037] (b) mixing the aqueous solution of step (a) with said
nucleic acid in an aqueous solvent, and
[0038] (c) reduce the volume of the product solution obtained in
step (b) to achieve a desired concentration of the said
composition.
[0039] It is yet another object of the present invention to provide
a method of administering a nucleic acid to a mammal, by
introduction of the composition, of the invention, into the
mammal.
[0040] A further object of the present invention are the use of the
composition of the invention in the manufacture of a medicament for
phrophylactic or therapeutic treatment of a mammal, or in the
manufacture of a diagnostic agent for the use in in vitro or in
vivo diagnostic methods.
[0041] These and other objects of the invention are provided by one
or more of the embodiments described below.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The composition according to the present invention can be
derived from cationic polysaccharide chitosan by the use of
chemical or enzymatic methods.
[0043] A preferred composition of the invention is wherein said
cationic oligomers contain preferably a weight fraction of less
than 20% of oligomers with DP<12 in addition to a weight
fraction of less than 20% with a DP>40 and most preferably a
weight fraction of less than 20% of oligomers with DP<15 in
addition to a weight fraction of less than 20% with a DP>30.
[0044] Compositions comprising complexes between low molecular
weight cationic chitosan oligomers and nucleic acid are described,
wherein the cationic chitosan oligomers have well-defined chain
lengths, narrow distribution of chain lengths and a well-defined
chemical composition. Typically, the cationic chitosan oligomer has
a molecular weight between 500 and 10,000 Da, preferably between
1,200 and 5,000 Da and most preferably between 3,000 and 4,700 Da.
Typically the cationic chitosan oligomer has a fraction of A-units
(F.sub.A) of 0-0.35 (65-100% de-N-acetylated units), preferably
between 0-0.1 (90-100% de-N-acetylated units) and most preferably
between 0-0.01 (99-100% de-N-acetylated units). Suitably, said
nucleic acid comprises a coding sequence that will express its
function when said nucleic acid is introduced into a host cell.
[0045] According to one embodiment of the invention, said oligomers
are derived from cationic polysaccharide chitosans followed by
fractionating a polydisperse oligomer pool into oligomers having
well-defined chain lengths, narrow distribution of chain lengths
and a fraction of A-units (F.sub.A) of 0-0.35 (65-100%
de-N-acetylated units), preferably between 0-0.1 (90-100%
de-N-acetylated units) and most preferably between 0-0.01 (99-100%
de-N-acetylated units). Typically, said oligomers consist of 6-50
monomer units, preferably of 10-30 monomer units and most
preferably of 15-25 monomer units, having a molecular weight
between 3,000 and 4,700 Da, and a FA of less than 0.01 (more than
99% de-N-acetylated units).
[0046] According to another embodiment of the composition of the
invention, said nucleic acid is selected from the group consisting
of RNA and DNA molecules. These RNA and DNA molecules can be
comprised of circular molecules, linear molecules or a mixture of
both. Preferably, said nucleic acid is comprised of plasmid
DNA.
[0047] According to a preferred embodiment of the present
invention, said nucleic acid comprises a coding sequence that will
express its function when said nucleic acid is introduced into a
host cell. For instance it can encode a biologically active
product, such as a protein, polypeptide or a peptide having
therapeutic, diagnostic, immunogenic, or antigenic activity.
[0048] The present invention is also concerned with compositions as
described above wherein said nucleic acid comprises a coding
sequence encoding a protein, an enzyme, a polypeptide antigen or a
polypeptide hormone or wherein said nucleic acid comprises a
nucleotide sequence that functions as an antisense molecule, such
as RNA.
[0049] Preferably the composition of the invention has a pH range
between 3.5 and 8.
[0050] The composition of the invention can also preferably be
derivatized with targeting ligands and/or stabilizing agents.
[0051] A further aspect of the invention is related to the liquid
droplet size of said composition after nebulization. Preferably,
the droplet size of the composition of the invention is essentially
equal to the droplet size of naked pDNA after nebulization.
[0052] The present invention is also directed to a method for
preparing the present composition, said method comprising the steps
of: providing the present cationic chitosan oligomer as described
above, (a) exposing said cationic chitosan oligomers to an aqueous
solvent in the pH range 3.5-8.0, (b) mixing the aqueous solution of
step (a) with said nucleic acid in an aqueous solvent and (c)
dehydrating the product solution obtained in step (b) to achieve a
high concentration of the composition before administration in
vivo. Step (c) can be obtained by (1) evaporating the liquid of the
product solution in step (b) to obtain the desired concentration,
or (2) lyophilize the product solution in step (b) followed by
reconstitution of the lyophilizate to obtain the desired
concentration of the composition. Typically, said nucleic acid is
present at a concentration of 1 ng/ml-300 .mu.g/ml, preferably 1
.mu.g/ml-100 .mu.g/ml and most preferably 10-50 .mu.g/ml in step
(b) and 10 ng/ml-3,000 .mu.g/ml, preferably 10 .mu.g/ml-1,000
.mu.g/ml and most preferably 100-500 .mu.g/ml in step (c) using the
evaporating method (1).
[0053] It should be understood, that a person skilled in the art
can form the present composition at different amine/phosphate
charge ratios to include negative, neutral or positive charge
ratios. However a preferred embodiment is wherein the composition
of the invention has a net positive charge.
[0054] The present invention is further concerned with a method of
administering nucleic acid to a mammal, using the composition of
the present invention, and introducing the composition into the
mammal. Preferably, said composition is introduced into the mammal
by administration to mucosal tissues by pulmonary, nasal, oral,
buccal, sublingual, rectal, or vaginal routes. According to another
preferred embodiment, said composition is introduced into the
mammal by parenteral administration such as intravenous,
intramuscular, intradermal, subcutaneous or intracardiac
administration.
[0055] The present invention is also concerned with use of the
composition of the invention in the manufacture of a medicament for
prophylactic or therapeutic treatment of a mammal, or in the
manufacture of a diagnostic agent for the use in in vivo or in
vitro diagnostic methods, and specifically in the manufacture of a
medicament for use in gene therapy, antisense therapy or genetic
vaccination for prophylactic or therapeutic treatment of
malignancies, autoimmune diseases, inherited disorders, pathogenic
infections and other pathological diseases.
[0056] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and specific examples, while indicating preferred
embodiments of the invention, are given by the way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
DESCRIPTION OF THE DRAWINGS
[0057] FIG. 1 shows the chemical composition of chitosan, where a
fragment of the chitosan chain contains one residue of
N-acetyl-.beta.-D-glucosami- ne (A-unit) and 3 residues of
.beta.-D-glucosamine (D-units). The amino group of the D-units may
be on a protonated or unprotonated form depending on pH.
[0058] FIG. 2a shows the chemical structure which is obtained after
depolymerisation of a chitosan by acid or by a chitosanase. Acids
cleave preferentially the glycosidic bond following an A-unit
(A-unit at the newly formed reducing end). Enzymes vary in their
specificities by hydrolysing both kinds of residues.
[0059] FIG. 2B shows the depolymerisation of chitosan by nitric
acid, which only attacks D-residues, which are rearranged to form
2,5-anhydro-D-mannose.
[0060] FIG. 3 shows results where a commercial chitosan with a
weight average molecular weight (M.sub.w) of 180.000 was analysed
by size-exclusion chromatography using a refractive index detector
and a multi-angle laser light scattering detector. Columns: TSK
G6000PWXL, 5000PWXL and 4000 PWXL (serially connected). Eluent: 0.2
M ammonium acetate, pH 4.5. RI detector: Optomed DSP (Wyatt). Light
scattering detector: DAWN DSP (Wyatt). Processing parameters (Astra
software v. 4.70.07): dn/dc=0.142 ml/g (determined off-line for
chitosan acetate, the value was found to be independent of
F.sub.A). A.sub.2: 5.0.multidot.10.sup.-3
mol.multidot.ml.multidot.g.sup.-2.
[0061] 3A: Elution profile, that is refractive index detector
signal, which is proportional to the concentration of chitosan,
combined with a plot of the calculated molecular weight in this
case expressed as chitosan in the acetate salt form as a function
of the elution volume.
[0062] 3B: The cumulative molecular weight distribution calculated
from the data given in 3A.
[0063] FIG. 4 shows SEC-MALLS chromatograms (4A), and differential
(4B) and cumulative (4C) molecular weight distributions of a
chitosan, which has been depolymerised by nitrous acid to obtain
different weight average molecular weights in the range from 41.500
to 13.400. Experimental conditions were the same as in FIG. 3.
[0064] FIG. 5: Calculated cumulative (A) and differential (B)
molecular weight distributions corresponding to the Kuhn
distribution for chitosan depolymerised to obtain 100, 50, 20 and
10 residues (DP.sub.n).
[0065] FIG. 6: Size-exclusion chromatograms of a fully
de-N-acetylated chitosan (F.sub.A<0.001) which has been
depolymerized by a) nitrous acid and reduced with NaBH.sub.4
(N-1-N4) or b) chitosanase (E1-E4) (Superdex 30; two 2.5.times.100
cm columns in series, eluent: 0.15M ammonium acetate, pH 4.5, flow
rate 0.8 ml/min). DP=6 indicates the elution volume of a fully
de-N-acetylated chitosan hexamer.
[0066] FIG. 7: SEC-MALLS chromatograms (7A) of a fully
de-N-acetylated chitosan (F.sub.A<0.001), which has been
depolymerised by nitrous acid and reduced with NaBH.sub.4
(un-fractionated sample) and fractions N1-N4 obtained as described
in FIG. 6. The experimental conditions were the same as in FIG. 3
except that a single column (TSK G3000 PWXL) was used. FIG. 7B
shows the corresponding cumulative molecular weight distributions
calculated from the data given in 7A.
[0067] FIG. 8: SEC-MALLS chromatograms (8A) of a chitosan, which
has been depolymerised by a chitosanase (un-fractionated sample)
and fractions E1-E4 obtained as described in FIG. 6. The
experimental conditions were the same as in FIG. 3 except that a
single column (TSK G3000 PWXL) was used. FIG. 8B shows the
corresponding cumulative molecular weight distributions calculated
from the data given in 7A.
[0068] FIG. 9 shows in vivo lung luciferase expression (pg/mg) 3
days after intra-tracheal administration of 25 .mu.g pLuc in mice
(four animals per group). Complexes between chitosan oligomers and
pLuc were prepared at an amine/phosphate charge ratio of 60:1
(+/-). The significantly highest luciferase expression was obtained
with pLuc complexed with the chitosan oligomer N0 having 18 as the
number average degree of polymerisation, as determined by
.sup.13C-NMR-spectroscopy. Statistical differences between mean
values were investigated using ANOVA. Differences between group
means were considered significant at P<0.05.
[0069] FIG. 10 shows in vivo lung luciferase expression (pg/mg) 3
days after intra-tracheal administration of 25 .mu.g pLuc in mice
(four animals per group). The chitosan oligomer N0 having 18 as the
number average degree of polymerization was fractionated into four
different samples having well-defined and narrow distributions of
their degrees of polymerization. Complexes between chitosan
oligomers and pLuc were prepared at an amine/phosphate charge ratio
of 60:1 (+/-). Complexes based on the fraction containing oligomers
having chain lenghts between 15-21 monomer units (N3), showed
significantly (p<0.05) higher gene expression compared to
complexes based on the unfractionated sample N0 having 18 as the
number average degree of polymerization. Statistical differences
between mean values were investigated using ANOVA. Differences
between group means were considered significant at P<0.05.
[0070] FIG. 11 shows results of the agarose gel retardation assay.
Complexes between chitosan oligomers and pLuc were prepared at an
amine/phosphate charge ratio of 60:1 (+/-). With increasing
molecular weight (degree of polymerization) of the chitosan
oligomer, a higher stability of formed complexes was observed.
Thus, complete retention of pLuc was detected with complexes formed
with the fraction containing 36-50 monomer units (N1) as compared
to complexes formed with 15-21 monomer units (N3).
[0071] FIG. 12 shows the luciferase gene expression in vitro after
incubating 293 cells with two batches of fractionated low molecular
weight cationic chitosan oligomers (N1 and E1) prepared 9 months
apart and commercial chitosan (Protasan UPG 210) ordered 3 years
apart, respectively. The gene expression varied 10-fold between the
two batches of Protasan UPG 210 complexed with pLuc at an
amine/phosphate charge ratio of 2.4:1 (+/-) but not significantly
between the two batches of fractionated low molecular weight
cationic chitosan oligomers (N1 and E1) complexed with pLuc at an
amine/phosphate charge ratio of 10:1 (+/-). Statistical differences
between mean values were investigated using ANOVA. Differences
between group means were considered significant at P<0.05.
[0072] FIG. 13 shows the liquid droplet size (mass median diameter,
MMD) after aerosolization of compositions containing cations
complexed with pLuc. Fractions of chitosan oligomers containing
15-21 (N3) and 36-50 (N1) monomer units and an ultra pure chitosan,
Protasan UPG 210 (UPC), were complexed with pLuc at an
amine/phosphate charge ratio of 60:1 (+/-) and 3:1 (+/-),
respectively. The MMD was clearly dependent on the composition. The
smallest droplet size was obtained with naked pLuc and the
composition containing 15-21 monomer units (N3) complexed with
pLuc. Statistical differences between mean values were investigated
using ANOVA. Differences between group means were considered
significant at P<0.05.
[0073] Using the expression of a reporter protein, luciferase, as a
model for a therapeutic protein in an in vivo lung model, it was
found that formulations comprising plasmid DNA and a certain
composition of chitosan oligomers having well-defined chain
lenghts, distribution of chain lenghts, and chemical composition,
are advantageous to achieve delivery of the nucleic acid into cells
of a selected tissue and to obtain in vivo expression of the
desired molecules encoded for by the nucleic acids.
[0074] It was found that a chitosan oligomer fraction, prepared
from chitosan, having a number-average degree of polymerization of
18 (DP.sub.n=18, as determined by .sup.13C-NMR-spectroscopy),
showing a relatively narrow size distribution as compared to the
Kuhn-distribution and having more than 99% D-units
(F.sub.A<0.01), formed stable complexes (as revealed by agarose
gel electrophoresis) with pLuc at an amine/phosphate charge ratio
of 60:1 (+/-). A significantly higher in vivo lung luciferase gene
expression was obtained with the polydisperse DP.sub.n=18 sample
compared to monodisperse chitosan oligomers having 6, 10 and 12
monomer units that formed unstable complexes with pLuc at an
amine/phophate charge ratio of 60:1 (+/-). The fact that stable
complexes resulted in a higher gene expression than unstable
complexes is in agreement with the prior art (Fischer et al., 1999;
Gebhart and Kabanov, 2001; Koping-Hoggard et al., 2001). However, a
decrease in luciferase expression was detected with stable
complexes formed with chitosan oligomers having higher average
molecular sizes than the DP.sub.n18 sample. The fraction DP.sub.n18
was further fractionated into fractions having more narrow
distributions that is 10-14 monomer units (N4), 15-21 monomer units
(N3), 22-35 monomer units (N2) and 36-50 monomer units (N1).
Complexes between the fraction having 15-21 monomer units and pLuc
resulted unexpectedly in the highest in vivo lung gene expression
although unstable complexes were formed at an amine/phosphate
charge ratio of 60:1 (+/-). The fraction 10-14 monomer units also
formed unstable complexes with pLuc and resulted only in a modest
luciferase expression.
[0075] Also, aerosolisation of complexes between the fraction
having 15-21 monomer units and pLuc resulted in comparable droplet
sizes as an aerosolised solution of naked pLuc. In contrast,
aerosolisation of the fraction having 36-50 monomer units complexed
with pLuc and UPC (approximately 1000-mer) complexed with pLuc
resulted in a 2 and 3-fold higher droplet size, respectively. This
formulation-dependent effect on the droplet size might be explained
by an increased viscosity of the solution with increasing molecular
weight of the cation, thus producing droplets of a larger size.
EXAMPLES
Example 1
Preparation of Low-Molecular Weight Chitosans
[0076] Chitosan Protasan UP G 210 (F.sub.A=0.17, weight-average
molecular weight of 162,000) was obtained from Pronova Biomedical
AS, Oslo, Norway. The low-molecular weight oligomer of
N-glucosamine was obtained by chemical depolymerisation of chitosan
using NaNO.sub.2 and subsequent reduction by NaBH.sub.4 as
described by T.o slashed.mmeraas et al., 2001, where the molecular
weight was controlled by the amount of NaNO.sub.2 relative to the
amount of chitosan. The fraction of acetylated units was controlled
by heterogeneous deacetylation to obtain FA of less than 0.001 as
determined by proton NMR-spectroscopy as described previously
(V.ang.rum et al., 1991). Typically, 1.0 gram of chitosan was
dissolved in 100 ml of 2.5% aqueous acetic acid, dissolved oxygen
was removed by bubbling nitrogen gas through the solution for 5
minutes, and 5 ml of a freshly prepared solution of NaNO.sub.2 in
distilled water (10 mg/ml) was added. The reaction was allowed to
proceed for 4 hours in darkness, whereafter the depolymerized
chitosan was conventionally reduced by adding 3 grams of NaBH.sub.4
overnight in darkness. The pH was adjusted to 4.5 using acetic
acid. The solution was dialysed (Medicell dialysis tubing, MWCO
12000-14000) three times against 0.2M NaCl and six times against
distilled water and lyophilized, to obtain the low-molecular weight
oligomer as their hydrochloride salt. Alternatively, the
low-molecular weight oligomers were obtained by enzymatic
depolymerisation using a chitosanase from Streptomyceus griseus
(Sigma C 9830 or Sigma C 0794) where the molecular weight is
controlled by the amount of enzyme relative to the amount of
chitosan and the incubation time. 0.5 gram of chitosan was
dissolved at a concentration of 20 mg/ml in 0.1 M
sodium-acetate/acetic acid buffer (pH 5.5) and 0.65 units of
Chitosanase (Sigma C 0794) was added to the chitosan solution and
incubated for 18 hours at 37.degree. C. The enzyme reaction was
stopped by decreasing the pH to 2 and then boiled for 5 minutes.
The depolymerized chitosan was dialysed and lyophilized as
described above, to obtain the low molecular weight enzyme-degraded
chitosan as their hydrochloride salts.
Example 2
Preparation and Characterization of Fractionated Samples
[0077] The low-molecular weight chitosans prepared as described in
Example 1 were fractionated by size-exclusion chromatography on two
2.5.times.100 cm columns connected in series as described
previously (T.o slashed.mmeraas et al., 2001). Fractions of 4 mL
were collected and pooled according to the chromatograms shown in
FIG. 6 (a and b), to obtain 4 fractions differing in molecular
weight designated
[0078] N1 (nitrous acid degraded) or E1 (chitosanase degraded)
[0079] N2 (nitrous acid degraded) or E2 (chitosanase degraded)
[0080] N3 (nitrous acid degraded) or E3 (chitosanase degraded)
[0081] N4 (nitrous acid degraded) or E4 (chitosanase degraded)
2TABLE 1 The samples were analyzed by SEC-MALLS, which yielded the
following chain length distributions (average of 3 injections):
Samples DP.sub.w DP.sub.n DP.sub.w/DP.sub.n Unfractionated N0 31 25
1.22 (nitrous acid degraded) N1 44 40 1.09 N2 27 26 1.03 N3 20 19
1.03 N4 14 13 1.04 Unfractionated E0 27 21 1.31 (chitosanase
degraded) E1 50 44 1.12 E2 33 30 1.06 E3 25 23 1.03 E4 17 16 1.07
wherein DPw = weight average DP and DPn = number average DP
Example 3
Formulation and In Vivo Lung Gene Expression
[0082] A polydisperse cationic chitosan oligomer fraction having a
degree of polymerization (DP) between 6-50 (number-average DP of 18
as determined from the non-reducing ends in the
.sup.13C-nmr-spectrum, N0) and well-defined cationic oligomers,
having DP's of 6, 10, 12, 10-14 (N4), 15-21 (N3), 22-35 (N2), 36-50
(N1) were prepared from chitosan according to the methods described
in Example 1 and Example 2. Firefly luciferase plasmid DNA (pLuc)
was purchased from Aldevron, Fargo, N. Dak., USA. Stock solutions
of cationic chitosan oligomers (2 mg/ml) were prepared in sterile
distilled deionized water, pH 6.2.+-.0.1 followed by sterile
filtration. Complexes between cationic chitosan oligomers and pLuc
were formulated at a charge ratio of 60:1 (+/-) by adding cationic
oligomer and then pLuc to sterile water under intense stirring on a
vortex mixer (Heidolph REAX 2000, KEBO Lab, Sp.ang.nga, Sweden).
After 15 min the complexes were concentrated by mild evaporation
under vacuum in a SpeedVac Plus centrifuge (Savant Instruments,
Holbrook, N.Y.) for approximately 90 min to obtain pLuc
concentrations of around 250 .mu.g/ml) (Koping-Hoggard et al.,
2001). In addition, pLuc was formulated with PEI 25 kDa (Aldrich
Sweden, Stockholm, Sweden) and an ultra pure chitosan, Protasan UPG
210 (Pronova Biopolymer, Oslo, Norway) at previously optimized
conditions, charge ratio 5:1 (+/-) and 3:1 (+/-) respectively
(Bragonzi et al., 2000; Koping-Hoggard et al., 2001).
[0083] Mice (male Balb/c, 6-8 weeks old, 4 animals per group,
Charles River, Uppsala, Sweden) were anesthesized with
ketamin/xylazine (5/20 vol %, 0.1 m/10 g of body weight), and the
trachea was surgically exposed with a 0.5 cm long skin incision in
the neck. 100 .mu.l of the complexes described aboved was slowly
administrated dropvise into the trachea and the mice were sutured.
At 72 h after administration, the animals were sacrified by carbon
dioxide and the lungs were surgically removed, washed in PBS and
0.3 ml ice-cold luciferase lysis buffer (Promega, Madison, Wis.)
with a protease inhibitor coctail (Complete, Boehringer Mannheim
Scandinavia AB, Bromma, Sweden) was added. The tissue samples were
quickly frozen in liquid nitrogen and stored at -80.degree. C.
until analysis.
[0084] In a cold room, the tissue samples were homogenized in a
bead beater (Biospec Products, Inc., OK) followed by centrifugation
(Centrifuge 5403, Eppendorf-Nethelar-Hinze GmbH, Hamburg, Germany)
at 4.degree. C. and 15,000 rpm for 10 min. An amount of 50 .mu.l of
the clear supernantant from each test tube was mixed with 50 .mu.l
of luciferase reagent (Promega) and analyzed by a luminometer
(Mediators PhL, Vienna, Austria) with an integration time of 8 s.
In order to quantify the luciferase expression, a standard curve of
luciferase (Sigma, St. Louise, Mo.) was prepared by adding defined
amounts of the luciferase standard to the supernatants of
homogenized tissues from untreated control animals. The total
protein content in each sample was analyzed by the BCA assay
(Pierce, Rockford, Ill.) and quantified using BSA (bovine serum
albumin) as a reference protein. The absorbance was measured at 540
nm on a microplate reader (Multiscan MCC/340, Labsystems Oy,
Helsinki, Finland).
[0085] Results of the gene transfection efficiency in mouse lungs
72 h after administration of pLuc complexed with cationic chitosan
oligomers of various degree of polymerization (molecular weight)
are shown in FIG. 9. Surprisingly, the significantly highest
luciferase expression was obtained with pLuc complexed with a
chitosan oligomer N0 having 18 as the number average degree of
polymerization.
[0086] The results of the gene transfection efficiency in mouse
lungs 72 h after administration of pLuc complexed with cationic
chitosan oligomers of various degree of polymerization (molecular
weight) are shown in FIG. 10. The chitosan oligomer N0 having 18 as
the number average degree of polymerization was fractionated, as
described in Example 2, into four samples having well-defined and
narrow distributions of their degrees of polymerization.
Surprisingly, the fraction containing chitosan oligomers having
chain lenghts between 15-21 monomer units (N3), showed higher gene
expression than PEI and significantly higher gene expression
compared to the un-fractionated sample N0 having 18 as the number
average degree of polymerization.
[0087] The results of the agarose gel retardation assay are shown
in FIG. 11. With increasing molecular weight (degree of
polymerization) of the chitosan oligomer, the stability of formed
complexes increases. Thus, almost complete retention of pLuc was
detected with complexes formed with the fractions containing 22-35
(N2) and 36-50 (N1) monomer units as compared to complexes formed
with 10-14 (N4) and 15-21 (N3) monomer units. The unfractionated
sample N0 having 18 as the number average degree of polymerization
also formed stable complexes with pDNA. A higher in vivo gene
expression (FIG. 10) was surprisingly obtained with the less stable
15-21 (N3) complexes compared to the stable complexes formed with
DPn18 (N0).
Example 4
In Vitro Gene Expression
[0088] Two different batches of fractionated low molecular weight
cationic chitosan oligomers; N1 and E1, as described in example 2
and prepared 9 months apart, and commercial chitosan (Protasan UPG
210, batch 1: apparent viscosity of 70 mPas, batch 2: apparent
viscosity of 146 mPas) ordered 3 years apart were complexed with
pLuc at charge ratios of 10:1 (+/-) and 2.4:1 (+/-), respectively,
as described in Example 2. Stable pDNA complexes were used.
[0089] 24 h before transfection, the epithelial human embryonic
kidney cell line 293 (ATCC, Rockville, Md., USA) were seeded at 70%
confluence in 96-well tissue culture plates (Costar, Cambridge,
UK). Prior to transfection, the cells were washed and then 50 .mu.l
(corresponding to 0.33 .mu.g pLuc) of the polyplex formulations was
added per well. After 5 h incubation, the formulations were removed
and 0.2 ml of fresh culture medium was added. The medium was
changed every second day for experiments exceeding two days. At 96
h and 144 h, cells were washed with PBS (pH 7.4), lysed (Promega)
and luciferase gene expression was measured with a luminometer
(Mediators PhL). The amount of luciferase expressed was determined
from a standard curve prepared with firefly luciferase (Sigma) and
total cell protein was determined using the bichinchoninic acid
test (Pierce).
[0090] The results of the luciferase gene expression in vitro after
incubating 293 cells with two batches of fractionated low molecular
weight cationic chitosan oligomers; N1 and E1 and commercial
chitosan Protasan UPG 210, respectively, are shown in FIG. 12. The
gene expression varied 10-fold between the two batches of Protasan
UPG 210 but not significantly between the two batches of the
fractionated low molecular weight cationic chitosan oligomers N1
and E1.
Example 5
Droplet Size After Aerosilisation
[0091] Complexes between cationic chitosan oligomers and pLuc were
prepared as described in Example 3 to obtain pLuc concentrations of
500 .mu.g/ml. As a control, an ultra pure chitosan (UPC, degree of
polymerization around 1000) complexed with pLuc were used at
optimal conditions, charge ratio 3:1 (+/-) (Koping-Hoggard et al.,
2001). Aerosols containing complexes between cationic chitosan
oligomers and pLuc were produced with the use of a nebulization
catheter (Trudell Medical International, London Ontario, Canada)
containing liquid- and gas (air)-channels. Firstly, 10011 of the
complex solution was loaded into a liquid reservoir coupled to the
nebulization catheter (liquid inlet). Then, to obtain aerosols,
pulses of pressurized air (3.5 bar) was applied for short time
periods over the liquid reservoir (20 ms) and the gas channels of
the nebulization catheter (50 ms). The droplet size of produced
aerosols was measured with a Mastersizer X (Malvern instruments
Ltd., Malvern, UK).
[0092] The liquid droplet size (mass median diameter, MMD) after
aerosolisation of compositions containing cations complexed with
pLuc are shown in FIG. 13. The MMD was clearly dependent on the
composition. The smallest droplet size was obtained with "naked"
pLuc and the composition containing 15-21 monomer units (N3)
complexed with pLuc.
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