EP1793810A1

EP1793810A1 - Nanoparticles and method for the production thereof

Info

Publication number: EP1793810A1
Application number: EP05783457A
Authority: EP
Inventors: Michael Ahlers; Conrad Coester; Klaus Zwiorek; Jan Zillies
Original assignee: Gelita AG
Current assignee: Gelita AG
Priority date: 2004-08-20
Filing date: 2005-08-18
Publication date: 2007-06-13
Also published as: NO20071458L; BRPI0514524A; IL180954A0; MX2007001996A; KR20070046850A; US20080003292A1; JP2008510688A; NZ551326A; CN1988892A; CA2575407A1; WO2006021367A1; DE102004041340A1; AU2005276675A1

Abstract

The aim of the invention is to provide biodegradable nanoparticles which ensure a uniform and definable transport of active substances, and to provide an appropriate method for producing these nanoparticles. To these ends, the invention provides that the nanoparticles are essentially comprised of an aqueous gelatin gel. The average diameter of the nanoparticles does not exceed 350 nm, the polydispersity index of the nanoparticles is less than or equal to 0.15, and a gelatin is used as a starting material for the production method whose proportion of gelatin with regard to the total gelatin is no greater than 40 % by weight with a molecular weight less than 65 kDa.

Description

Nanoparticles and process for their preparation

The present patent application relates to nanoparticles, the use of nanoparticles for the production of medicaments and a method for the production of nanoparticles.

Nanoparticles as carrier systems for pharmaceutical substances have been known since the 1970s. They allow a targeted transport of the active ingredients in a ge desired area of the body, wherein the release takes place only at the destination (so-called drug delivery systems). At the same time, the active ingredient, which has not yet been released, is effectively shielded against metabolic influences of the body. Thus, side effects can be minimized by the Wirkstoff¬ molecules predominantly and targeted arrive at their actual site of action and less burden on the whole organism.

For the production of nanoparticles, numerous synthetic starting materials, such as e.g. Polyacrylates, polyamides, polystyrenes and cyanoacrylates described. However, the decisive disadvantage of these macromolecules lies in their poor or absent biodegradability.

Fibronectin, various polysaccharides, albumin, collagen and gelatin are known, among other things, as natural, degradable carrier materials. Another difficulty with the known nanoparticles consists in the sometimes wide size distribution, which is disadvantageous in terms of a uniform release and transport behavior. Although the size distribution of such nanoparticles can be made narrower to a certain extent by complicated centrifugation and other separation methods, this does not lead to a satisfying result.

The object underlying the present application is therefore to provide biodegradable nanoparticles which ensure uniform and definable drug delivery. At the same time, the object is to specify a suitable method for producing this nanoparticle.

This object is achieved with the nanoparticles of the type mentioned in that they consist essentially of an aqueous gelatin gel, wherein the average diameter of the nanoparticles is at most 350 nm and the polydispersity index of the nanoparticles is less than or equal to 0.15.

Gelatine has as starting material! for nanoparticles a number of advantages. It is available in defined composition and purity and has a relatively low antigenic potential. Gelatine is also approved for parenteral use, including as a plasma expander.

In addition, the amino acid side chains of gelatin offer the simple possibility of chemically modifying the surface of the nanoparticles, of crosslinking the latins or of covalently binding active ingredient molecules to the particles.

For the purposes of the present application, the term "aqueous gelatin gel" is to be understood as meaning that the gelatin contained in the nanoparticles in hydrated form, ie as hydrocolloid. Since the nanoparticles are always surrounded by an aqueous solution during their preparation and use, all information on the size and polydispersity of the nanoparticles relates to this hydrated form. The determination of these parameters is carried out using the standard method of photon correlation spectroscopy (PCS), which is described in more detail below.

For the purposes of the invention, the expression "consisting essentially of" is to be understood as meaning that the nanoparticles are at least 95% by weight or more, preferably 97% by weight or more, even more preferably up to 98% by weight or more and most preferably 99% by weight or more of the aqueous gelatin gel.

The polydispersity index is a measure of the size distribution of the nanoparticles, whereby theoretically values between 1 (maximum scattering) and 0 (identical size of all particles) are possible. The low polydispersity index of the nanoparticles according to the invention of not more than 0.15 ensures targeted and controllable drug delivery as well as the release of the drug at the desired target site, in particular when uptake of the nanoparticles by body cells.

Particular preference is given to nanoparticles having a polydispersity index of less than or equal to 0.1.

The size of the nanoparticles is a decisive factor for their applicability and may vary depending on the field of application. In many cases, nanoparticles with an average diameter of at most 200 nm are preferred. Another embodiment of the invention relates to nanoparticles having an average diameter of at most 150 nm, preferably from 80 to 150 nm. These can be used by utilizing the so-called EPR effect (enhanced permeability and retention). This effect makes it possible to specifically treat tumor cells which have a higher uptake rate than nanoparticles of the stated size range than healthy cells.

Another parameter for the size distribution of the nanoparticles is the band width of the diameter, which is preferably at a maximum of 20 nm above and below the mean value. The bandwidth can also be determined by means of PCS.

The properties of the nanoparticles according to the invention can also be influenced by the molecular weight distribution of the gelatin contained. Important in this context is the proportion of low molecular weight gelatin, in particular the proportion of gelatin having a molecular weight below 65 kDa, based on the total gelatin contained in the nanoparticles. This proportion is preferably below 40 wt .-%. Particularly advantageous is a proportion of less than 30 wt .-%, preferably 20 wt .-% and Weni¬ ger.

In the state of the art, in connection with gelatine, nanoparticles are usually described which, in addition, contain further structural polymers (for example nanoparticles produced by the coacervation process, as described in WO 01/47501 A1). The nanoparticles made of pure gelatin prepared hitherto are either unstable or do not have the parameters described above with regard to particle diameter and size distribution which are advantageous for selective drug delivery. In a further preferred embodiment, the gelatin contained in the nanoparticles is crosslinked. Networking will increase the stability of the nanoparticles! In addition, the degree of decomposition of the nanoparticles can be deliberately adjusted by the degree of crosslinking selected. This is advantageous since different application areas usually require defined degradation times of the nanoparticles.

In particular in the case of crosslinking, it is important that the proportion of gelatin having a molecular weight below 65 kDa is less than 20% by weight.

Non-crosslinked nanoparticles are suitable for extracorporeal, in particular diagnostic, applications in which, below the melting point of gelatin, e.g. can be operated at room temperature.

In contrast, the above-described crosslinked nanoparticles are particularly suitable for therapeutic applications.

The gelatin may be chemically crosslinked, e.g. by formaldehyde, dialdehyde, isocyanates, diisocyanates, carbodiimides or alkyldihaiogenides.

Alternatively, enzymatic crosslinking, e.g. by transglutaminase or laccase.

In a further embodiment, the nanoparticles according to the invention are dried, preferably up to a water content of not more than 15% by weight. Another embodiment of the invention relates to nanoparticles, on the surface of which a pharmaceutical active substance is bound.

In a preferred embodiment, prior to the binding of the drug, the surface of the nanoparticles is chemically modified, e.g. by the reaction of free amino or carboxyl groups of the gelatin, whereby charged Seiten¬ chains or side chains with a new chemical functionality entste¬ hen.

The binding of the pharmaceutical active substance to the nanoparticles or to the chemically modified nanoparticles can be effected by adsorption forces, by covalent bonds or by ionic bonds. For example, nanoparticles whose surfaces are positively charged by a corresponding chemical modification, DNA or RNA fragments are ionically bound ge¬.

In a further embodiment, the binding of the active ingredient to the nanoparticles takes place via a spacer.

If they are crosslinked, the nanoparticles described above can be used according to the invention for the production of medicaments.

Particularly advantageous is the use of the nanoparticles for intracellular drug delivery systems, in particular as a carrier for nucleic acids or peptides.

Medicaments containing nanoparticles according to the invention can preferably be used in gene therapy. The present invention further relates to a process for the preparation of nanoparticles of the type described above.

According to the invention, the object underlying the invention with regard to the method is achieved by using a gelatin as the starting material for the preparation process whose maximum amount of gelatin having a molecular weight of less than 65 kDa, based on the total gelatin 40 wt .-% is.

By using such a gelatin, nanoparticles having a low polydispersity and bandwidth of the particle diameter can be produced in a simple manner, in particular the nanoparticles according to the invention having a polydispersity index of less than or equal to 0.15.

In the method according to the invention, an aqueous solution is first prepared from such a gelatin, the pH of which is then adjusted to a value below 7.0. By adding a suitable precipitating agent to this solution, the dissolved gelatin is desolvated in the form of nanoparticles, which are then separated from the solution by simple centrifugation. Fractionation of the nanoparticles, e.g. by gradient centrifugation, is not necessary, since its polydispersity is already in a sufficiently low range as a result of the production process according to the invention.

An addition of excipients to the aqueous gelatin solution, in particular of salts or surface-active substances such as detergents, is not required in the context of the method according to the invention. Nanoparticles according to the invention are therefore preferably substantially free of the stated additives. The inventive method thus enables the production of Nanoparticles, which essentially consist only of an aqueous gelatin gel.

The use of gelatin with the molecular weight distribution described above ensures the formation of stable nanoparticles. Gelatins with a higher low molecular weight fraction in this process increasingly lead to the formation of larger aggregates or unstable particles.

Preferably, the proportion of gelatin having a molecular weight of less than 65 kDa is at most 30% by weight, most preferably at most 20% by weight.

In a preferred embodiment of the method, the adjusted pH of the gelatin solution is less than or equal to 3.0, preferably in the range of 1.5 to 3.0. Within this interval, the pH can be z.T. an influence on the mean particle size are exerted, with a lower pH tends to result in smaller nanoparticles.

In a further preferred embodiment acetone, alcohols, e.g. Ethanol, or mixtures of these precipitants used in succession or with water, acetone is be¬ preferred as the precipitating agent.

The use of volatile precipitants of this kind largely avoids the fact that fractions of the precipitant are incorporated into the nanoparticles and / or remain so that they consist essentially only of the aqueous gelatin gel. For the preparation of crosslinked nanoparticles, after addition of the precipitating agent and before centrifuging, the addition of a crosslinking agent takes place. In this embodiment, the proportion of gelatin having a molecular weight below 65 kDa is preferably 20% by weight or less in order to counteract agglomeration of the particles upon crosslinking. With this method it is possible to produce very uniform nanoparticles with a maximum bandwidth of ± 20 nm and a polydispersity index of not more than 0.1.

In the following the invention will be explained in more detail with reference to the examples with reference to the drawings. They show in detail:

FIG. 1 shows the gel permeation chromatograms of two gelatins (FIGS. 1A and 1B, respectively) representing the molecular weight distribution of the respective gelatin;

FIG. 2: an electron micrograph of nanoparticles according to the invention; and

FIG. 3 shows a size distribution of nanoparticles produced according to the invention.

Determination of molecular weight distribution

As described above, the molecular weight distribution of the gelatin can be used to influence the properties of the nanoparticles produced therefrom. The molecular weight distribution can be determined by gel permeation chromatography (GPC). The determination is carried out on an HPLC system with the following components:

HPLC pump: Pharmacia 2249 UV detector: truck 2151

Separation column: TFK 400 SWXL with precolumn (Tosoh Biosep GmbH)

Plasticizer: 1% by weight SDS, 100 mmol / l Na ₂ SO ₄ ,

10 mmol / l NaH ₂ PO ₄ / NaOH pH 5.3

It is a 1 wt .-% gelatin solution in water by 30 minutes swelling of the gelatin and then dissolving at about 60 ⁰ C. After filtration through a 0.2 μl disposable filter, 30 μl of the gelatin solution are mixed with 600 μl of flow agent and 30 μl of a 0.01% strength by weight benzoic acid solution. The GPC is carried out with 20 μl of this mixture at a flow rate of 0.5 ml / min and UV detection at 214 nm.

The assignment between elution volume and molecular weight is carried out by calibrating the system with a standard gelatin having a known molecular weight distribution. By dividing the chromatogram into defined ranges and integrating the UV detector signal, the proportion of gelatin which lies in the respective molecular weight range can be calculated.

FIG. 1 shows by way of example the gel permeation chromatograms of two different gelatins:

FIG. 1A shows the GPC of a commercial pork rind gelatin (Type A gelatin) with a bloom value of 175. Due to the high proportion of gelatin with a molecular weight below 65 kDa, which is above 45% by weight, this gelatin is for the production process according to the invention are not suitable for nanoparticles and leads to particles with too high polydispersity or agglomeration of the particles.

FIG. 1B shows the GPC of a pigskin gelatin having a bloom value of 310 and a proportion of gelatin having a molecular weight of less than 65 kDa of about 15% by weight. This gelatin is very well suited for the production process according to the invention.

Determination of the mean particle diameter and the polydispersity index

Photon correlation spectroscopy allows the determination of the mean particle diameter of the nanoparticles, the polydispersity index and the bandwidth of the particle diameter above and below the mean value.

The measurements were carried out with a BI-200 SM Goniometer Version 2 (Brookhaven Instruments Corp., Holtsville, NY, USA). For this purpose, nanoparticle suspensions were used with a concentration of 10 to 50 ug / ml in de-mineralized water.

example 1

This example describes the preparation of crosslinked nanoparticles from gelatin, the GPC of which is shown in FIG. 1B (with a proportion of gelatin having a molecular weight below 65 kDa of about 15% by weight).

300 mg of said gelatin are dissolved in water at 50 ⁰ C. After adjusting the pH to 2.5 with hydrochloric acid, the deoivatation of the crystals is carried out by the dropwise addition of 45 ml of acetone. After stirring for 10 minutes, 40 μl of an 8% strength aqueous glutaraldehyde solution are added. solution and then stirred for a further 30 min. The nanoparticles thus crosslinked are separated from the solution by centrifugation at 10,000 g for 10 minutes and purified by redispersion three times in acetone / water (30/70). After the last redispersing the acetone at 50 ⁰ C is evaporated.

This simple process leads to nanoparticles according to the invention without additional separation steps, for which an average particle diameter of about 160 nm was determined at a polydispersity index of about 0.08 using the PCS method described above. The distribution of the nanoparticles by size classes is shown graphically in FIG.

Comparative experiments on non-crosslinked nanoparticles have shown that the proportion of low molecular weight gelatin in the nanoparticles produced largely corresponds to the proportion in the starting material.

Example 2

Crosslinked nanoparticles are prepared as described in Example 1, with a pigskin gelatin having a bloom value of 270, whose proportion of gelatin having a molecular weight below 65 kDa being about 19% by weight, being used as starting material.

For the directly obtained nanoparticles according to the invention, an average particle diameter of about 173 nm and a polydispersity index of about 0.08 were determined by the PCS method described above. The size distribution was comparable to the nanoparticles prepared according to Example 1.

Claims

PATENT APPLICATIONS

1. Nanoparticles, consisting essentially of an aqueous gelatin gel, wherein the average diameter of the nanoparticles is at most 350 nm and the polydispersity index of the nanoparticles is less than or equal to 0.15.

2. Nanoparticle according to claim 1, wherein the polydispersity index of the particle Nano¬ less than ^'or equal to 0.1.

3. nanoparticles according to claim 1 or 2, wherein the average diameter of the nanoparticles is at most 200 nm.

4. Nanoparticle according to one of claims 1 to 3, wherein the average diameter of the nanoparticles is at most 150 nm.

5. nanoparticle according to one of claims 1 to 4, wherein the bandwidth of the diameter of the nanoparticles at a maximum of 20 nm above and below the half of the mean value.

6. nanoparticles according to one of claims 1 to 5, wherein the proportion of Ge latin having a molecular weight below 65 kDa, based on the total contained in the nanoparticles gelatin, a maximum of 40 wt .-% is.

7. Nanoparticles according to any one of claims 1 to 5, wherein the proportion of Ge latins having a molecular weight below 65 kDa, based on the total contained in the nanoparticles gelatin, a maximum of 30 wt .-% is.

8. Nanoparticle according to one of claims 1 to 5, wherein the proportion of Ge latin having a molecular weight below 65 kDa, based on the total contained in the nanoparticles gelatin, a maximum of 20 wt .-% is.

9. nanoparticles according to one of claims 1 to 8, wherein the gelatin contained in the nanoparticles is crosslinked.

10. nanoparticles according to claim 9, wherein the gelatin is crosslinked by means of formaldehyde, dialdehydes, isocyanates, diisocyanates, carbodiimides or Alkyldiha- logeniden.

11. Nanoparticles according to claim 9, wherein the gelatin is enzymatically crosslinked.

12. Nanoparticles according to claim 11, wherein the gelatin is crosslinked by means of transglutaminase or laccase.

13. Nanoparticle according to one of claims 1 to 12, wherein the Wasserge¬ content of the nanoparticles is at most 15 wt .-%.

14. Nanoparticles according to one of claims 1 to 13, wherein a pharmaceutical active substance is bound to the surface of the nanoparticles.

15. Nanoparticles according to claim 14, wherein the binding of the pharmaceutically active substance is effected by a chemical modification of the surface of the nanoparticles.

16. nanoparticles according to claim 14 or 15, wherein the pharmaceutically active substance is adsorptively bound.

17. Nanoparticles according to claim 14 or 15, wherein the pharmaceutically active substance is covalently bound.

18. A nanoparticle according to claim 14 or 15, wherein the pharmaceutically active substance is ionically bound.

19. Nanoparticles according to one of claims 14 to 18, wherein the pharmaceutic agent is bound via a spacer.

20. Use of nanoparticles according to any one of claims 1 to 19 as a biodegradable carrier for the manufacture of a medicament.

21. Use according to claim 20, wherein the nanoparticles are part of an intracellular drug delivery system, in particular as a carrier for nucleic acids or peptides.

22. Use according to claim 20 or 21, wherein the medicament is a medicament for gene therapy.

23. A process for the preparation of nanoparticles consisting essentially of an aqueous gelatin gel, comprising the following steps: a) Preparation of an aqueous Geiatinelösung, wherein the proportion of Ge latins having a molecular weight of less than 65 kDa, based on the total gelatin, a maximum of 40 wt .-% is;

b) adjusting the pH of the gelatin solution to below 7.0;

c) precipitation of the gelatin by addition of a precipitant; and

d) Separation of the nanoparticles by centrifugation.

24. The method according to claim 23, wherein in step a) the proportion of gelatin having a molecular weight of less than 65 kDa, based on the total gelatin, a maximum of 30 wt .-% is.

25. The method according to claim 23, wherein in step a) the proportion of gelatin having a molecular weight of less than 65 kDa, based on the total gelatin ge, a maximum of 20 wt .-% is.

26. The method according to any one of claims 23 to 25, wherein in step b) the pH is adjusted to a value less than or equal to 3.0.

27. The method of claim 26, wherein in step b) the pH is adjusted to a value in the range of 1.5 to 3.0.

28. The method according to any one of claims 23 to 27, wherein in step c) the precipitant acetone, an alcohol or a mixture of both, if appropriate gege¬ in aqueous solution.

29. The method according to any one of claims 23 to 28, wherein between the steps c) and d) a step

c ') adding a crosslinking agent to the gelatin solution

he follows.

30. The method of claim 29, wherein the crosslinking agent is selected from formaldehyde, dialdehydes, isocyanates, diisocyanates, carbodiimides or alkyl dihalides.

The method of claim 29, wherein the crosslinking agent is an enzyme.

The method of claim 31, wherein the crosslinking agent is laccase or transglutaminase.