CN100586990C - Multifunctional dendrimers and hyperbranched polymers as drug and gene delivery systems - Google Patents

Abstract

The present invention relates to the synthesis of multifunctional dendrimeric and hyperbranched polymers for use as drug delivery systems and gene delivery systems (carriers of genetic material) for biologically active drug compounds, the latter by condensation with genetic material. In particular, the present invention relates to the synthesis of multifunctional compounds based on suitable dendrimeric or hyperbranched polymers having introduced on their surface functional groups X, Y, Z. In addition, for gene delivery to cells, these multifunctional systems will become cationic systems for forming complexes with negatively charged genetic material. The functional group allows the delivery system to be recognized by a complementary cellular receptor. Furthermore, they make the system stable in the biological environment and facilitate its transport through the cell membrane.

Description

Multifunctional dendrimers and hyperbranched polymers as drug and gene delivery systems

technical field

The present invention relates to the synthesis of multifunctional dendrimers and hyperbranched polymers, especially but not exclusively to the modification of their surface end groups so that they can be used as effective drug and gene delivery systems.

Background technique

The structural features of dendrimers and hyperbranched polymers (dendrimers) and especially the presence of nanocavities in their interior or the presence of several groups on their outer surface make these polymers extremely useful as pharmaceuticals and Candidates for gene delivery applications. Bioactive pharmaceutical compounds can be encapsulated within nanocavities, while surface groups can be appropriately modified, allowing the preparation of multifunctional dendrimers.

Recently the application of dendrimers as drug carriers has been studied and functional dendrimers have been prepared. These dendrimers encapsulate bioactive drug molecules within their nanocavities. This is due to the hydrophobic or in some other cases hydrophilic environment inside the nanocavity which can encapsulate either hydrophilic or hydrophobic compounds, respectively. As mentioned above, the structural features of the dendrimers make possible the controlled release of the incorporated bioactive compounds. However, it has been difficult to prepare multifunctional dendrimers that simultaneously exhibit all the desired properties in order to effectively act as drug carriers, and in particular exhibit biocompatibility and biodegradation Sexuality, for long-term circulation in the human body, biostable, for attachment to cell receptors, with target ligands, and controlled release properties of encapsulated bioactive compounds. The absence of one of the above properties renders the drug carrier ineffective. Therefore, commercialization of several biologically active drug compounds is not possible if the drug carriers used do not exhibit the multifunctional features described above.

In general therapeutics, viral vectors are widely used as carriers of genetic material. Although viral vectors are generally effective, they cause problems for the health of patients. To this end, synthetic vectors, such as non-viral vectors for genetic material, have recently been introduced. Liposomes and dendrimers, for example, have gained much attention for their use in gene therapy because of their safety profile compared to viral vectors. In particular, synthetic non-viral vectors for genetic material present little risk of genetic recombination within the genome.

Transfection with synthetic non-viral vectors is also characterized by low cytotoxicity, high reproducibility and ease of use.

However, currently known synthetic vectors have disadvantages due to their generally low effectiveness compared to viral vectors and their inability to be used for the expression of target genes. Specifically, for efficient gene expression, the gene must be transferred inside the nucleus, and the procedure must overcome a series of intracellular and extracellular obstacles. These obstacles include targeting of cells, efficient delivery of vectors and their genetic material across the cell membrane, and the need to release vectors from endosomes following pinocytosis.

With regard to the synthetic vectors described in the literature, some or all of these problems are solved, however, the desired end goal is not achieved. The object of the present invention is to simultaneously solve or overcome all of the above-mentioned problems by introducing suitable functional groups on the surface of dendrimers or hyperbranched polymers. The problems described above call for the development of new and efficient vectors for the delivery of genetic material to the nucleus. Specifically, these vectors should simultaneously possess targeting ability, exhibit stability in biological systems, have the ability to efficiently transport through the cell membrane together with the linked genetic material, and the latter complex can be released from the nucleus after pinocytosis. released in the body.

Such stable and efficient synthetic gene carriers can be dendrimers or hyperbranched polymers. In contrast to liposomes, which are often unstable, dendrimers and hyperbranched polymers are available as stable nanoparticles, the size of the dendrimers depending on their generation, and the variety of functional groups that can be conveniently introduced on their surface. The properties decisively affect its performance and thus its application.

Contents of the invention

The object of the present invention is to prepare polyfunctional dendritic polymers which can be used as effective drug carriers for biologically active pharmaceutical compounds and genetic material. Preferred dendrimers include symmetric dendrimers and asymmetric hyperbranched polymers. By employing these polyfunctional dendrimers and hyperbranched polymers (dendrimers), commercialization of drug compounds that are not commercially possible with conventional carriers is made possible. Alternatively, genes can be transferred to cells for gene therapy.

Hyperbranched polymers are not widely used as drug carriers.

Their applications are of great interest due to their ease and low cost of preparation compared to dendrimers.

The end groups of dendritic and hyperbranched polymers can be suitably modified in order to become multifunctional and to make it possible to encapsulate pharmaceutical compounds within their nanocavities.

Suitably selected structural features of dendritic and hyperbranched polymers render these molecules biocompatible and biodegradable at the same time.

In addition, suitable targeting ligands can be carried for attachment to cellular receptors, and the molecules can exhibit biostability for prolonged circulation in biological fluids. Controlled release of the encapsulated pharmaceutical compound may be permitted.

When these polymers are positively charged on their surface, they can form complexes upon interaction with oligonucleotides or DNA.

The present invention relates to the preparation of polyfunctional dendrimers which, in addition to their positively charged surfaces, lead to the formation of complexes with negatively charged DNA, which may also carry functional groups, as follows As mentioned, this facilitates the delivery of genetic material.

The proposed structural features of these polymers especially for biomedical applications are as follows: a. Presence of functional groups on the surface of dendritic or hyperbranched polymers. These functional groups can be introduced in stages. b. Depending on their nano-environment, there are nano-cavities inside polymers that can encapsulate various compounds, this latter property making compounds particularly useful as drug carriers. c. The presence of cationic charges within these polymers is required when used for gene delivery as they will interact with negatively charged DNA leading to the formation of various complexes. The complex thus formed can be introduced by pinocytosis into the nucleus for gene therapy.

According to the present invention, there are provided dendritic polymers and asymmetric hyperbranched polymers with symmetrical chemical structures, characterized in that they are modified so as to exhibit: - at least one chemical bond capable of forming three or more chemical bonds Atoms of an element - bonded to said at least one atom are various terminal functional groups which in general a) have low toxicity or no toxicity at all, b) allow for complementary acceptability from cells molecules in the body that recognize said polymers, c) stabilize the polymers within the organism's biological environment, and d) facilitate transport of said polymers across cell membranes.

Preferably, the polymer is cationized for complex formation with DNA when said compound is to be a gene delivery system, eg a carrier for genetic material.

Conveniently, the polymer can be cationized by introducing ammonium, quaternary ammonium or guanidinium groups on the end groups of the dendrimers.

Advantageously, atoms of a chemical element capable of forming three or more chemical bonds can be nitrogen or other suitable characteristic groups, such as carbon or silicon.

Preferably, the modified dendrimers may be diaminobutane poly(propyleneimino) dendrimers (DAB), or other dendrimers of similar structure, such as PAMAM dendrimers.

Conveniently, the modified hyperbranched asymmetric polymers may be derived from polycondensation of anhydrides such as succinic anhydride, phthalic anhydride or tetrahydrophthalic anhydride with dialkylamines such as diisopropylamine.

Advantageously, modified hyperbranched asymmetric polymers can be derived from anionically polymerized epoxy derivatives and 1,1,1-tris(hydroxyalkyl)propane.

Conveniently, modified hyperbranched asymmetric polymers can be derived from anionically polymerized glycidol and 1,1,1-tris(hydroxymethyl)propane (PG-5).

Conveniently, the modified dendritic polymer or the modified hyperbranched asymmetric polymer may have on its surface functional groups, for example polyalkylene glycols and preferably poly(ethylene glycol), wherein said surface functional groups include molecular weight polymer chains.

Advantageously, the modified dendritic polymer or the modified hyperbranched asymmetric polymer may comprise functional groups comprising at least one group complementary to the receptor site of the cell, such as a guanidinium group, a carbohydrate Compounds (eg, mannose, glucose, galactose), folic acid, RGD receptors, bases (eg, adenine, thymine, guanidine, cytosine), or barbiturates.

Advantageously, the modified dendrimers or modified hyperbranched asymmetric polymers may include a compound that facilitates transport of the dendrimers or modified hyperbranched polymers together with any encapsulated active pharmaceutical ingredients or genes At least one group of material, such as a guanidinium moiety, an oligo- or polyarginine derivative, or a polypropylene oxide moiety.

Conveniently, the modified dendrimers or modified hyperbranched asymmetric polymers may comprise functional groups containing at least one targeting ligand, e.g. guanidinium groups, carbohydrates (e.g. mannose, glucose, galactose) , folic acid, RGD receptors, bases (eg, adenine, thymine, guanidine, cytosine), or barbiturates.

Preferably, the modified dendritic polymers and modified hyperbranched asymmetric polymers can be used as drug carriers for biologically active pharmaceutical compounds or for carrying genetic material.

Conveniently, the biologically active drug compound carried by the modified dendritic polymer or the modified hyperbranched asymmetric polymer may be betamethasone or a betamethasone derivative.

The present invention also provides methods for the synthesis of multifunctional dendrimers and hyperbranched polymers so that they can be used as drug carriers for bioactive pharmaceutical compounds, the method is characterized by the stepwise modification of the surface of these polymers, which includes: a. replace amino groups or other toxic groups on the surface with hydroxyl, carboxyl or quaternary ammonium groups, or other non-toxic groups, b. introduce various Polymer chains of different molecular weights (PEGylation) in order to protect the polymer from the organism's MPS (Mononuclear Phagocyte System), c. Introduction of recognizable groups complementary to receptors or tissues, i.e. guanidinium, carbohydrate Compound moieties (mannose, glucose, galactose), folate or RGD receptors, bases (adenine-thymine, guanidine-cytosine) or barbiturates in order to increase the targeting ability of the vector. d. Introducing groups that facilitate the transport of the carrier and encapsulated active pharmaceutical ingredient through cell membranes, such as guanidinium moieties, oligo- or polyarginine derivatives or polypropylene oxide moieties.

Preferably, the process comprises: carrying out an initial reaction of the external amino or hydroxyl groups of the dendritic or hyperbranched polymer with a suitable protected polymer bearing a reactive group at one end, such as isocyanate, ring Oxide or N-hydroxysuccinimide; followed by reaction of the largest part of the amino groups of the resulting polymer with ethyl isocyanate to replace the toxic amino groups; subsequent reaction of the previously obtained polymer to convert the amino groups into a recognizable group such as a guanidinium group; followed by the introduction of one or more groups that facilitate transport of the carrier across the cell membrane, such as polyarginine or propylene oxide chains.

Conveniently, the polymers are cationized to form complexes with DNA when the compounds are to be gene delivery systems, for example when they are to be carriers of genetic material.

Advantageously, the method is characterized in that, when the toxic groups on the surface are amino groups, small aliphatic chains having less than 8 carbon atoms, preferably 2 or 3 carbon atoms, can be introduced for substitution.

The present invention provides pharmaceutical formulations comprising biologically active pharmaceutical compounds or genetic material encapsulated within modified multifunctional dendrimers or modified multifunctional hyperbranched asymmetric polymers.

The present invention also provides a method for producing a pharmaceutical formulation for delivery of a biologically active pharmaceutical compound or genetic material, the method comprising: synthesizing a symmetrical dendrimer or an asymmetric hyperbranched polymer by modifying the surface of the polymer in stages Substances, including: a. Substituting amino groups or other toxic groups on the surface with hydroxyl, carboxyl or quaternary ammonium groups or other non-toxic groups, b. In dendritic carriers or hyperbranched polymers, such as poly(ethylene glycol) (PEG (L) polymer chains of various molecular weights are introduced on the surface in order to thus protect the polymer from the organism's MPS (Mononuclear Phagocyte System). c. Introduction of recognizable groups complementary to receptors or tissues, namely guanidinium groups, carbohydrate moieties (mannose, glucose, galactose), folic acid or RGD receptors, bases (adenine-thymine, guanidine- Cytosine) or barbiturates in order to improve the targeting ability of the vector. d. introducing groups that facilitate delivery of the carrier and encapsulated biologically active drug compound through cell membranes, such as guanidinium moieties, oligo- or polyarginine derivatives, or polypropylene oxide moieties; and using said The modified polymer encapsulates biologically active pharmaceutical compounds or genetic material.

Preferably, the polymer is cationized for complex formation with DNA, when the compound is to be a carrier of genetic material.

Conveniently, modified dendritic polymers or modified hyperbranched asymmetric polymers comprising encapsulated bioactive pharmaceutical compounds or loaded genetic material are used for therapeutic applications.

Advantageously, modified dendritic polymers or modified hyperbranched asymmetric polymers comprising encapsulated biologically active pharmaceutical compounds or carrying therapeutically useful genetic material are used for the manufacture of pharmaceutical dosage forms.

Conveniently, a modified dendritic polymer or a modified hyperbranched asymmetric polymer comprising an encapsulated biologically active pharmaceutical compound or loaded genetic material is used in the manufacture of a drug for the treatment of the same disease or condition as the compound or genetic material. drug.

Description of drawings

Figure 1 shows a molecule of general formula I with a symmetrical dendritic structure, the object of the present invention, wherein the symbol (.) can be an atom of a chemical element capable of forming 3 or more chemical bonds, such as nitrogen or a suitable characteristic group group, the straight line (-) corresponds to the aliphatic chain and the external functional groups X, Y, Z generally: a) allow recognition of the above-mentioned polymer molecule from the complementary receptors of the cell, b) allow the same polymer to be recognized in a biological environment Internal stabilization and c) groups that facilitate transport of these polymers across cell membranes.

Figures 2 and 3 show the molecular structures of two different asymmetric hyperbranched polymers (which are the object of the present invention), where the symbol (g) can be an atom of a chemical element capable of forming 3 or more chemical bonds , such as nitrogen or a suitable characteristic group, the straight line (-) corresponds to the aliphatic chain and the external functional groups X, Y, Z generally: a) allow the recognition of the above polymer molecule from the complementary receptors of the cell, b ) groups that provide stability to these polymers within the biological environment and c) which facilitate transport of these polymers across cell membranes.

Figure 4 shows the step-by-step introduction of functional groups on the surface of dendrites (or hyperbranched polymers) according to one embodiment of the present invention, that is: in the first step, external amino groups or hydroxyl groups in dendrites are combined with Suitable polymers with reactive groups such as epoxy or N-hydroxysuccinimide are reacted.

In a second step, the larger fraction of amino groups remaining on the surface of the dendrons is reacted, for example with ethyl isocyanate, to replace the toxic amino groups.

In the third step, a recognition group, such as a guanidinium group, is introduced.

In the fourth step, groups are introduced that facilitate the transfer of the carrier and encapsulated drug compound across the cell membrane, such as guanidinium groups, oligo-arginine or poly-arginine.

Figure 5 illustrates the formation of complexes between dendrimer and DNA or oligonucleotides and their transport across cell membranes.

Figure 6 shows a graph of the release of encapsulated betamethasone valerate as a function of the concentration of aqueous sodium chloride.

Figure 7 shows the introduction of functional groups on the surface of a hyperbranched polymer according to one embodiment of the present invention, that is, in a one-step reaction, the introduction of two functional groups attached to terminal hydroxyl groups, such as protected PEG chains and leaves Ester targeting ligands.

Detailed ways

In one embodiment, the present invention relates to the synthesis of multifunctional symmetrical dendrimers. These polyfunctional symmetrical dendrimers are listed by the general formula (I) shown in FIG. 1 . Such polymers may be, for example, diaminobutane poly(propyleneimino) dendrimers.

The invention also relates to the synthesis of polyfunctional asymmetric hyperbranched polymers. These polyfunctional asymmetric hyperbranched polymers are listed by general formula (II) shown in FIG. 2 and hyperbranched polymers of formula (III) in FIG. 3 .

Such asymmetric polymers are obtained, for example, from the polycondensation of succinic anhydride, phthalic anhydride or tetrahydrophthalic anhydride with diisopropylamine or from the anionic polymerization of glycidol with 1,1,1-tris Polymers obtained from (hydroxymethyl)propane.

In formulas I, II and III, the symbol (y) is an atom of a chemical element that can form three or more chemical bonds, such as nitrogen or other suitable characteristic groups, such as tertiary amino groups, and the straight line (-) corresponds to lipid The family chain and the external functional groups X, Y, Z generally can: a) allow the molecule of the above polymer to be recognized from the cell's complementary receptors, b) stabilize the above polymer in the biological environment, and c) contribute to transport of these polymers across cell membranes.

Those characteristic structural features which make the polymers according to the invention particularly useful for biomedical applications are the following: a) the presence of functional characteristic groups on the surface of dendrimers or hyperbranched polymers, these functional characteristic groups Agglomerates result from stepwise introduction on the polymer surface, eg as shown in Figure 4, and b) depending on its nanoenvironment, there are nanocavities inside the polymer that can encapsulate various compounds.

Modification of the surface of dendritic or hyperbranched polymers with the introduction of positive charges in the first stage (molecularly engineered surfaces of dendritic or hyperbranched polymers) makes the polymer suitable for binding negatively charged genetic material (DNA , plasmids, oligonucleotides). The dendritic or hyperbranched polymer carrier-gene material complex thus formed is finally introduced into the nucleus for gene therapy by pinocytosis.

For the preparation of such polyfunctional dendritic and hyperbranched polymers, which are the object of the present invention, commercially available dendrimers are used, for example from the company DSM and sold under the names DAB-32 and DAB-64. Its structure is modified by gradually introducing functional groups in a suitable reactor and under suitable experimental conditions. Figure 4 shows a reaction scheme for the synthesis of eg multifunctional dendritic drug delivery systems.

In another embodiment of the invention, PAMAM can be used identically instead of DAB in a suitable reactor.

In the present invention, biologically active compounds can be introduced primarily inside the nanocavities of dendrimers or hyperbranched polymers, while introducing on their outer surfaces suitable functional groups aimed at forming nanoscale carriers, which in general They are characterized by the fact that they have low or no toxicity, that they are stable within the biological environment and that they possess targeting and delivery capabilities towards specific cells.

When using dendrimers or hyperbranched polymers as suitable carriers of genetic material (for gene delivery), for example by introducing ammonium groups, quaternary ammonium or guanidinium ions at the end groups of the dendrimers or hyperbranched polymers, A positive charge is thereby introduced for binding negatively charged genetic material (DNA, plasmids, oligonucleotides), as described below.

Subsequently, various functional groups are introduced on the surface of dendrimers or hyperbranched polymers, with the ultimate aim of transporting genetic material within the nucleus. Specifically, non-toxic dendrimers or hyperbranched polymers are chosen, or the starting compounds are modified in order to make them non-toxic and biocompatible.

Subsequently, functional groups are introduced which: i) stabilize the DNA-carrier complexes within the biological environment, ii) provide targeting properties to specific cells or tissues, iii) facilitate their transport across membranes, and iv) Has the ability to be released from endosomes following pinocytosis.

The thus formed complexes of dendrimers or hyperbranched polymers with genetic material can eventually be introduced into cells by pinocytosis. The genetic material ends up in the nucleus for gene therapy through intercellular processes.

All of these properties are achieved according to the method described below, wherein the external end groups of the dendrimer or hyperbranched polymer are suitably modified (in a suitable series of reactions according to well-known methods of synthetic organic chemistry, Molecular design of dendrimers or hyperbranched polymers) in order to achieve: a) replacement of toxic end groups, such as amino groups, with non-toxic, such as hydroxyl, carboxyl or quaternary ammonium groups, b) Polymer chains of various molecular weights, such as poly(ethylene glycol) (PEGylated), are incorporated on the surface. The polymer is thus protected from the organic MPS (Mononuclear Phagocyte System). c. Introduction of recognizable groups complementary to the cell's receptors, such as guanidinium groups, carbohydrate moieties (mannose, glucose, galactose), folic acid or RGD receptors, nucleobase moieties (adenine, thymine, guanidine , cytosine) or barbiturate groups in order to improve the targeting ability of the carrier. d. Introducing groups that facilitate the transport of the carrier and the encapsulated active pharmaceutical ingredient or gene across the cell membrane, such as guanidinium moieties, oligo- or polyarginine derivatives or polypropylene oxide moieties. Positively charged moieties such as ammonium, quaternary ammonium, guanidinium can be introduced for complex formation with genetic material (DNA, plasmids, oligonucleotides).

Synthesis of such polyfunctional dendrimers can be achieved by using commercially available dendrimers or hyperbranched polymers. Figure 4 shows an illustrative example demonstrating the procedure for the synthesis of multifunctional dendrimers.

Initially combine the external amino or hydroxyl groups of dendrimers or hyperbranched polymers with poly(ethylene glycol) polymers of selected molecular weights bearing reactive groups such as isocyanate, epoxide or N-hydroxysuccinimide moieties reaction. After this first step, the majority of the remaining amino groups in the resulting dendrons are reacted, for example with ethyl isocyanate, to reduce the presence of toxic primary amino groups on the outer surface. In a third step, the last remaining primary amino group can be transferred to a targeting group, such as a guanidinium group. In a further stage, groups that facilitate the transport of the drug carrier as well as the encapsulated active ingredient through the cell membrane, such as oligo-arginine or poly-arginine moieties, can be introduced. In the context of the present invention, the guanidinium group introduced as a targeting ligand can facilitate the delivery of the delivery system encapsulating the active pharmaceutical ingredient through the cell membrane. Cationization of dendrimers or hyperbranched polymers is required for attachment of negatively charged genetic material to the dendrimers for the formation of various stable complexes with the genetic material to be transferred to cells.

The reactions described above can be carried out in aqueous media at room temperature. Purification of the product can be performed by dialysis by passing the by-product through a semi-permeable membrane.

Typical dendrimers or hyperbranched polymers that can be used in the present invention are, for example, symmetrical diaminobutane poly(propyleneimino) dendrimers or asymmetric hyperbranched polymers, e.g. formed from polycondensed succinic anhydride , a polymer obtained by phthalic anhydride or tetrahydrophthalic anhydride and diisopropylamine, or a polymer obtained by anionic polymerization of glycidol and 1,1,1-tris(methylol)propane.

Polymers that can be used as protective coatings for dendrimers are, for example, polyethylene glycols of various molecular weights with reactive groups, such as isocyanates, that react with dendrimers or hyperbranched polymers , epoxide or N-hydroxysuccinimide moieties, for example using isocyanate derivatives of methoxypoly(ethylene glycol) with an average molecular weight of 5000.

Substitution or reaction of toxic groups such as amino groups can be achieved by reaction with alkyl isocyanates or alkyl epoxides. The latter converts primary amino groups into secondary amino alcohols. In the present invention, ethyl isocyanate is preferred because it reacts conveniently with primary amino groups.

Furthermore, for the introduction of targeting ligands (guanidinium groups in the examples described above), 1H-pyrazolo-1-carboxamidine hydrochloride can be used to convert the dendrimer in question The exogenous amino group is converted into this group. The guanidinium group as well as the oligo- and poly-arginine moieties facilitate transport of the vector across cell membranes. For gene delivery applications, Figure 5 illustrates the preparation of complexes and their delivery.

Examples of the use of dendrimers as drug carriers were performed using lipophilic bioactive compounds that are completely insoluble in water, such as betamethasone valerate. These compounds were found to solubilize up to 14.5% inside the multifunctional dendrimers.

They are protected by poly(ethylene glycol) chains (PEG), and they have, as targeting ligands, guanidinium groups that allow targeting of the polymer to cell or tissue receptors. It was also established that betamethasone valerate remained encapsulated within these multifunctional dendrimers even in acidic environments. However, upon addition of aqueous NaCl, bioactive corticosteroid compounds were released from the nanocavities of the dendrimers (Fig. 6).

Due to the structural features that dendrimers share with similar multifunctional hyperbranched polymers, it is strongly expected that the latter polymers will show similar or almost identical behavior as drug carriers to those derived from multifunctional dendrimers and performance. Figure 7 shows the reaction scheme for the synthesis of multifunctional hyperbranched polymers based on commercially available polymers, such as PG-s5.

In this specification, the amounts mentioned in the following examples are in moles, unless otherwise stated.

Example

Materials and Methods

The 4th and 5th generation diaminobutane poly(propyleneimine) dendrites (in the following schemes -DAB-32 and DAB-64 are represented by No.1) having 32 and 64 amino groups respectively on the outer surface , DSM Fine Chemicals) were used as starting materials.

Methoxy poly(ethylene glycol) isocyanate (represented as No. 2 in the following scheme - MW5000, Shearwater Polymers, INC), ethyl isocyanate (Aldrich) and 1H-pyrazole-1-carboxamidino hydrochloride Salt (Fluka) (designated as No. 3 in the scheme below) was used for polyfunctionalization of the dendrimers.

Betamethasone valerate (indicated as No. 4 in the scheme below), which is a lipophilic drug, was supplied by EFFECHEM S.R.L. of Italy, and it was used for encapsulation and release studies.

Glycidyltrimethylammonium chloride (represented as No. 5 in the scheme below) and folic acid (represented as No. 6 in the scheme below) were purchased from Flika.

Hyperbranched polyether polyols (in the scheme below - MW5000, PG5 denoted No. 7) were purchased from Hyperpolymers GmbH and used after lipophilization.

The dendrimers and basic organic starting materials described above are shown in the following schemes.

Multifunctionalization Example 1 of Process A Dendrimer

Step 1. Diaminopoly(propyleneimino) dendron 0.001mol (it is the 5th generation commercially available product) (or any other generation product) and 0.004mol molecular weight is 5000 methoxypoly(ethylene dimethoxy) alcohol) isocyanate dissolved in water. A small amount of triethylamine aqueous solution was added to the obtained solution to obtain a solution with pH=13. The solution was stirred at room temperature for several hours. The solution was subsequently purified by dialysis through a semi-permeable membrane for 24 hours in order to remove all small molecular weight impurities from the reaction mixture. The incorporation of poly(ethylene glycol) moieties from step 1 within the dendrimer was determined using NMR spectroscopy.

¹ H NMR δ=6.20 and 5.90 (s, NHCONH), 3.55 (s, OCH ₂ CH ₂ O), 3.25 (s, OCH ₃ ), 3.15 (m, CH ₂ NHCONHCH ₂ ), 2.70 (m, CH ₂ NH _{2 ) , 2.45} ( _m , _NCH2CH2CH2N , _{NCH2CH2CH2CH2N , NCH2CH2CH2NH2 , NCH2CH2CH2NH ) , 1.55 ( m} , _NCH2CH2 CH ₂ N, NCH ₂ CH ₂ CH ₂ CH ₂ N, NCH ₂ CH ₂ CH ₂ NH), 1.42 (NH ₂ ).

¹³ C NMR δ=159.7 (NHCONH), 71.5 (OCH ₂ CH ₂ O), 58.5 (OCH ₃ ), 53.5 (NCH ₂ CH ₂ CH ₂ N, NCH ₂ CH ₂ CH ₂ CH ₂ N), 51.2 (NCH ₂ CH ₂ CH ₂ NH ₂ ), 50.5 (NCH ₂ CH ₂ CH ₂ NHCO), 43.5 (NHCONHCH ₂ CH ₂ ), 42.4 (NCH ₂ CH ₂ CH ₂ NHCO), 39.5 (CH ₂ NH ₂ ), 30.4 (CH ₂ CH ₂ NH ₂ ), 27.9 (NCH ₂ CH ₂ CH ₂ NHCO), 24.8 (NCH ₂ CH ₂ CH ₂ N, NCH ₂ CH ₂ CH ₂ CH ₂ N).

Step 2. Add 0.052 mol ethyl isocyanate also dissolved in water to 0.001 mol I dissolved in water. The pH of the solution was adjusted to 13 by adding 40% trimethylamine in water. The mixture was reacted at room temperature for several hours, dialyzed with a 12400 cut-off membrane to remove low molecular weight compounds, and finally lyophilized to provide compound II. The functionalization of the second step was established by H and NMR.

¹ H NMR (500 MHz, DMSO-d ₆ ) δ=6.05 (broad s, NHCONH), 3.50 (s, OCH ₂ CH ₂ O), 3.25 (s, OCH ₃ ), 3.05 (m, CH ₂ NHCONHCH ₂ ), 2.70 (m, CH ₂ NH ₂ ), 2.35 (m, NCH ₂ CH ₂ CH ₂ N, NCH ₂ CH ₂ CH ₂ CH ₂ N, NCH ₂ CH ₂ CH ₂ NH ₂ , NCH ₂ CH ₂ CH ₂ NH), 1.45(m, _{NCH2CH2CH2N , NCH2CH2CH2CH2N , NCH2CH2CH2NH} ) _{, 1.35 ( NH2 )} , 0.98(t, _CH3 ) _.

¹³ C NMR (62.9 MHz, D ₂ O) δ = 159.7 (NHCONH), 71.5 (OCH ₂ CH ₂ O), 58.5 (OCH ₃ ), 53.5 (NCH ₂ CH ₂ CH ₂ N, NCH ₂ CH ₂ CH ₂ CH ₂ N), 51.2 (NCH ₂ CH ₂ CH ₂ NH ₂ ), 50.5 (NCH ₂ CH ₂ CH ₂ NHCO), 43.5 (NHCONHCH ₂ CH ₂ O), 42.4 (NCH ₂ CH ₂ CH ₂ NHCO), 39.5 (CH ₂ NH ₂ ), 37.8 (NHCONHCH ₂ CH ₃ ), 30.4 (CH ₂ CH ₂ NH ₂ ), 27.9 (NCH ₂ CH ₂ CH ₂ NHCO), 24.8 (NCH ₂ CH ₂ CH ₂ N, NCH ₂ CH ₂ CH ₂ CH ₂ N), 14.8 (CH ₃ ).

Step 3. To 0.001 mol of the dendrimer prepared in step 1 dissolved in dry DMF was added 0.01 mol of 1H-pyrazole-1-carboxamidino hydrochloride and 0.01 mol of diisopropyl, also dissolved in dry DMF Ethylamine. The reaction mixture was allowed to react overnight at room temperature, and the resulting product was precipitated with diethyl ether and centrifuged. The solid compound was dissolved in water and dialyzed with a 12400 cut-off membrane. The solvent is removed, and the remaining material is dried sufficiently to provide compound III. The introduction of the guanidinium group was established ^{by1H and13C} NMR.

¹ H NMR (500MHz, DMSO-d ₆ ) δ=7.65(broad s, NH of guanidinium group), 6.95(broad s, NH ₂ ⁺ ), 6.05(broad s, NHCONH), 3.50(s, OCH ₂ CH ₂ O), 3.25(s, OCH ₃ ), 3.05(m, CH ₂ NHCONHCH ₂ , NCH ₂ CH ₂ CH ₂ NHC(NH ₂ ) ₂ ⁺ ), 2.35(m, NCH ₂ CH ₂ CH ₂ N, NCH ₂ CH _{2CH2CH2N , NCH2CH2CH2NH )} , 1.45 _{( m , NCH2CH2CH2N , NCH2CH2CH2CH2N , NCH2CH2CH2NH )} , _{0.98 ( t} , CH ₃ ).

¹³ C NMR (62.9MHz, D ₂ O) δ = 159.7 (NHCONH), 157.2 (NHC (NH ₂ ) ₂ ⁺ ), 71.5 (OCH ₂ CH ₂ O), 58.5 (OCH ₃ ), 53.5 (NCH ₂ CH _{2 CH2N , NCH2CH2CH2CH2N ) ,} 50.5 _{( NCH2CH2CH2NHCO , NCH2CH2CH2NHC} ( _{NH2 ) 2+} ), _{43.5 ( NHCONHCH2CH2O} ), ^42.4 (NCH ₂ CH ₂ CH ₂ NHCO), 42.2 (NCH ₂ CH ₂ CH ₂ NHC(NH ₂ ) ₂ ⁺ ), 37.8 (NHCONHCH ₂ CH ₃ ), 28.2 (NCH ₂ CH ₂ CH ₂ NHC(NH ₂ ) ₂ ⁺ ), 27.9 (NCH ₂ CH ₂ CH ₂ NHCO), 24.8 (NCH ₂ CH ₂ CH ₂ N, NCH ₂ CH ₂ CH ₂ CH ₂ N), 14.8 (CH ₃ ).

Example 2

Step 1. Quaternization of diaminobutane poly(propyleneimine) dendrimers.

Partial quaternization of poly(propyleneimine) dendrimers was performed as follows: To a solution of 0.113 mmol DAB-32 (0.398 g) in 10 ml water was added 1.938 mmol glycidyltrimethylammonium chloride (260 μl ). The mixture was allowed to react overnight, then dialyzed against _H2O with a 1200 cut-off membrane to remove unreacted epoxide, and lyophilized. The incorporation of quaternary ammonium was established ^{by1H and13C} NMR spectra recorded in _D2O . Quaternization was evidenced by the appearance of the expected 4 new signals at 2.60, 3.16, 3.34 and 4.26 ppm in the ¹ H spectrum and at 55.1 , 56.9, 67.4 and 71.8 ppm in the ¹³ C NMR spectrum. Additionally, in the ¹³ C NMR spectrum, two new signals appeared at 28.0 and 49.5 ppm, corresponding to the α and β methylene carbons relative to the newly formed secondary amino group. According to the integral ratio of the signal at 3.16 ppm (corresponding to quaternary methyl protons) for the signal at 1.58 ppm (corresponding to all β-methylene protons attached to the tertiary, secondary and primary amino groups of the dendron), Estimated degree of substitution. The degree of substitution was found to be 33%.

Synthesis of Folate Active Esters

This is a non-commercially available organic intermediate, and it was required for the next step to prepare multifunctional dendrimers by the following procedure: 0.594 mmol of folic acid dissolved in 7.5 ml of anhydrous DMSO was mixed with 0.595mmol TEA (82.5pl) and 0.595mmol DCC (0.123g) in 1ml anhydrous solvent were reacted for 1 hour. To this mixture was added 0.594 mmol N-hydroxysuccinimide in 1 ml dry DMSO and allowed to react overnight under inert conditions.

DCU was recovered by filtration and precipitated in drying and collected by filtration. The active ester was vacuum dried for about 2 hours and then used to react with the quaternized DAB-32 obtained previously.

Step 2. Introducing folic acid onto the quaternized DAB-32

The previously prepared folic acid active ester was used as starting material for the introduction of folic acid targeting ligands into dendrimers according to the following procedure: 0.0137 mmol of quaternized DAB-32 in 7 ml of anhydrous DMSO was added to dissolve in 0.0413 mmol of folic acid-NHS active ester in 1 ml of the same dry solvent. After a period of 5 days, the product was precipitated in dry _Et2O , first dialyzed against pH 7.4 phosphate buffer, then dialyzed against _H2O with a membrane with a cut-off of 1200 and lyophilized.

^{Both1H and13C} NMR spectra were recorded in _D2O . By a characteristic signal at 8.6ppm (corresponding to the methine at position 7 of the pterin ring), and two doublets at 6.7 and 7.7ppm (corresponding to the aromatic protons of the benzyl ring moiety), thus prove the presence of folic acid. According to the signal at 8.6ppm (corresponding to the proton at position 7 of the pterin ring) and the signal at 4.54ppm (corresponding to the methine group in the hydroxyl group bearing the glycidyl reagent, which comes from the epoxy The integral ratio of ring opening) estimates the average number of folic acid molecules in each conjugate. The average number of folate residues in the dendrimer derivatives was estimated to be three. Furthermore, the folate content in these dendrimers was also determined by UV spectroscopy in PBS (7.4) using the extinction coefficient value ζ280=74620 M ⁻¹ cm ⁻¹ . These results were further confirmed ^by13C NMR spectroscopy. The end product is quaternized (introduction of cationic charge) and functionalized by targeting folate ligands, while its amino groups (primary, secondary and tertiary) can also be protonated in the biological environment, thereby exhibiting buffering capacity.

B. PEGylation of Hyperbranched Polymer Functionalized Polyglycerol PG-5

To a solution of 0.04094 mmol PG-5 in 10 ml water dissolved in aqueous trimethylamine pH 13.0, 1639 mmol methoxy poly(ethylene glycol) isocyanate dissolved in 10 ml water was added. The mixture was allowed to react under an inert atmosphere for about 4 hours, dialyzed with a membrane with a cut-off value of 12400 to remove unreacted polymer and PEG-isocyanate, and finally lyophilized and dried under vacuum to obtain PEGylated PG5.

¹ H and ¹³ C NMR spectra were recorded in D ₂ O. The signal at 3.32 ppm (corresponding to the terminal methyl group in this reagent), and the signal at 3.25 ppm (corresponding to the _CH2 proton relative to the amide bond ( _CONHCH2- )) evidence the introduction of the PEG moiety. The formation of PEGylated hyperbranched polymer polyols was also established ^by13C NMR spectroscopy. The degree of substitution was estimated from the integral ratio of the signal at 3.24 ppm (corresponding to _CH2 protons relative to the amide bond ( _CONHCH2- )) to the signal at 0.82 ppm (corresponding to methyl groups of the core moiety). The average number of m-PEG in each polymer was 2.

Synthesis of NH ₂ -PEG-folate

NH ₂ -PEG- folate. The reaction mixture was stirred overnight at room temperature in the dark. At the end of the reaction, double the volume of water was added, and the insoluble by-product, dicyclohexylurea, was filtered by centrifugation. The supernatant was then dialyzed against 5 mM NaHCO ₃ buffer (pH 9.0) and then against deionized water to remove unreacted folic acid from the mixture (cut-off 1200). Traces of unreacted polyoxyethylenebisamine were then removed by batch absorption on cellulose phosphate cation exchange resin prewashed with an excess of 5 mM phosphate buffer (pH 7.0). The product NH ₂ -PEG-folate was again dialyzed against water, lyophilized, and ¹ H and ¹³ C NMR spectra were recorded in D ₂ O. Within the product, in the ¹ H spectrum, a characteristic signal at 8.64 ppm (corresponding to the methine at position 7 of the pterin ring) and two doublets at 6.74 and 7.60 ppm (corresponding to the benzyl Aromatic protons in the base moiety), proving the presence of folic acid. The average number of folic acid molecules in each conjugate was estimated from the integral ratio of the signal at 8.64 ppm to the signal at 3.15 ppm (corresponding to the α-methylene group adjacent to the remaining amino groups). According to the ¹³ CNMR spectrum, only the y-carboxyl group in folic acid was reacted by replacing its α-methylene signal at 30.4 ppm with a new signal at 32.6 ppm.

Synthesis of PG5-PEG-folate

PG5-PEG-folate was synthesized by reacting polyglycerol PG-5 with excess succinic anhydride in DMF overnight at slightly elevated temperature in order to achieve 5-10% reaction of polyglycerol hydroxyl groups. The reaction product was dialyzed against water, and its structure was confirmed ^{by1H and13C} NMR experiments. In the ¹ H spectrum, two new signals appeared at 2.5 and 2.6 ppm, corresponding to the α- and 3-methylene groups in the newly formed ester bond, respectively. Additionally, amide bond formation was achieved by reacting NH2 _- PEG-folate with modified polyglycerol PG5 in dry DMF, and in the presence of dicyclohexylcarbodiimide and pyridine, as described above. The reaction product was dialyzed against water (5000 cut-off), and folate incorporation was confirmed again by ¹ H and ¹³ C NMR experiments. The presence of PEG-folate on the hyperbranched polymer was evidenced by a characteristic signal at 8.64 ppm in the ¹ H spectrum. The average number of folic acid molecules in each conjugate was estimated from the integral ratio of the signal at 8.64 ppm to the signal at 0.82 ppm (corresponding to the methyl group on the core group of the polymer). In addition, the intramolecular folate content of the conjugates was also determined by quantitative UV spectroscopy in PBS (pH 7.4) using the extinction coefficient value ζ280 = 74620 M ⁻¹ cm ⁻¹ .

Encapsulation and Release of Betamethasone Derivatives

Encapsulation of the betamethasone derivative in the polyfunctional dendrimers prepared in Example 1 was carried out by dissolving the dendrimer and the betamethasone valerate derivative in a mixture of chloroform/methanol. After distillation of the solvent, a thin film is obtained which is dispersible in water. Dendrimers with encapsulated compounds were in the aqueous phase, while non-encapsulated material remained insoluble in water and was removed by centrifugation. Table 1 gives the percentage of betamethasone valerate encapsulated within the multifunctional dendrimer. For comparison, data from encapsulated probes such as flowers, a well-known type, were included.

Table 1. Solubility comparison of perylene (PY) and betamethasone valerate (BV) in parent and multifunctional dendrimer

Release of, for example, betamethasone valerate was achieved with the dropwise addition of aqueous sodium chloride (Figure 6). It was observed that upon addition of 0.8M NaCl, the biologically active compound was almost completely released from the multifunctional dendrimers.

Preparation of multifunctional dendrimers carrying genetic material

Add positively charged polyfunctional dendrimers to plasmid DNA (3-7 mg) so that the dendrimers to DNA charge ratio is 3.5 in various media such as natural serum, 300 mM sodium chloride in water, RPMI-1640 :1 to 8.5:1.

The terms "comprising", "comprising" and variations thereof, when used in the specification and claims, mean that the specified features, steps, components or integers are included. The terms should not be interpreted as excluding the presence of other features, steps, components or integers.

The features disclosed in the foregoing description or the following claims or drawings (expressed in their specific form or in the form of means for achieving the disclosed function, or the method or process of achieving the disclosed result), may stand alone or in the form of Any combination of these features is used to realize various forms of the present invention.

Claims (23)

1. a dendritic polymer or the asymmetrical hyperbranched polymer with symmetric chemical structure is characterized in that, described polymkeric substance is through modification, thereby comprises: can form 3 or the atom of at least one chemical element of more a plurality of chemical bonds; Be bonded to the various terminal functionality on described at least one atom, described terminal functionality is whole: a) have low toxicity or do not have toxicity at all, b) make it possible to from the complementary acceptor of cell the molecule of the above-mentioned polymkeric substance of identification, c) make polymkeric substance stable in coenocorrelation, and d) help described polymkeric substance to carry by cytolemma.

2. according to the dendritic polymer or the asymmetrical hyperbranched polymer of claim 1, wherein this polymkeric substance of cationization is in order to form complex compound with DNA, when described polymkeric substance will become the carrier of genetic material.

3. according to the dendritic polymer or the asymmetrical hyperbranched polymer of claim 2, wherein by on the end group of this dendrimer, introducing ammonium, quaternary ammonium or guanidine radicals, thus this polymkeric substance of cationization.

4. according to the dendritic polymer or the asymmetrical hyperbranched polymer of claim 1, wherein can form 3 or the atom of the chemical element of more a plurality of chemical bonds be nitrogen, carbon or silicon.

5. according to the dendritic polymer or the asymmetrical hyperbranched polymer of claim 1, wherein said dendritic polymer is poly-(propylene imino) dendrimer of diaminobutane or polyamide-amide dendrimer.

6. according to the dendritic polymer or the asymmetrical hyperbranched polymer of claim 1, wherein hyperbranched polymer is derived from polycondensation acid anhydrides and two alkanamines.

7. according to the dendritic polymer or the asymmetrical hyperbranched polymer of claim 1, wherein hyperbranched polymer is derived from anionoid polymerization epoxide and 1,1,1-three hydroxyalkyl propane.

8. the dendritic polymer of claim 1 or asymmetrical hyperbranched polymer, wherein hyperbranched polymer is derived from anionoid polymerization Racemic glycidol and 1,1, the 1-TriMethylolPropane(TMP).

9. according to any one dendritic polymer or asymmetrical hyperbranched polymer of claim 1-8, wherein said various terminal functionality is included in the polymer chain of the lip-deep various molecular weight of dendritic polymer or hyperbranched polymer.

10. according to any one dendritic polymer or asymmetrical hyperbranched polymer of claim 1-8, wherein said various terminal functionality comprises at least one group of acceptor site complementary with cell, and this at least one group is guanidine radicals, carbohydrate, folic acid, RGD acceptor, base or barbiturate(s).

11. according to any one dendritic polymer or asymmetrical hyperbranched polymer of claim 1-8, wherein said various terminal functionality comprises that at least a helping carry dendritic polymer or the hyperbranched polymer of modification and the group of any active pharmaceutical ingredient of sealing or genetic material by cytolemma.

12. according to any one dendritic polymer or asymmetrical hyperbranched polymer of claim 1-8, wherein functional group comprises at least a target part, this target part is guanidine radicals, carbohydrate, folic acid, RGD acceptor, base or barbiturate(s).

13. according to any one dendritic polymer or asymmetrical hyperbranched polymer of claim 1-8, wherein, described polymeric encapsulate bioactive medical compounds or is being carried genetic material.

14. according to the dendritic polymer or the asymmetrical hyperbranched polymer of claim 13, wherein bioactive medical compounds is Betamethasone Valerate or Valisone.

15. according to claim 1-14 any one dendritic polymer or the synthetic method of hyperbranched polymer; it is characterized in that the surface of these polymkeric substance of modification stage by stage; comprising: a. hydroxyl; carboxyl or quaternary ammonium group; or other nontoxic group replaces lip-deep amino or other poisonous group; b. on the surface of dendritic polymer or hyperbranched polymer, introduce the polymer chain of various molecular weight; so that the protection polymkeric substance exempts from organic mononuclear phygocyte system and engulfs; but c. introducing and acceptor or organize the complementary recognition group; it is guanidine radicals; the carbohydrate part; folic acid or RGD acceptor; nuclear alkali part or barbiturate(s); so that improve the target ability of carrier; introducing helps by the group of cytolemma delivery vehicles with the active pharmaceutical compounds of sealing, i.e. guanidine part with d.; oligomerization arginine or poly arginine or poly(propylene oxide) part.

16. according to the synthetic method of claim 15, wherein carry out the initial action of the outside amino of dendrimer or hyperbranched polymer or hydroxyl and suitable protection polymkeric substance, described protection polymkeric substance at one end has reactive group; Carry out the amino of largest portion of resulting polymers and the reaction of ethyl isocyanate subsequently, in order to replace deleterious amino; Make front resulting polymers reaction subsequently, but in order to amino is transformed into recognition group; Introduce the one or more groups that help by the cytolemma delivery vehicles subsequently.

17. the synthetic method according to claim 15 or 16 is characterized in that, the described polymkeric substance of cationization is in order to form complex compound with DNA.

18. the synthetic method according to claim 15 or 16 is characterized in that, when the poisonous group on surface is amino, introduces the aliphatic chain have less than 8 carbon atoms for replacement.

19. a pharmaceutical preparation, it is characterized in that it comprise be encapsulated in claim 1-14 any one dendritic polymer or asymmetrical hyperbranched polymer in bioactive medical compounds or genetic material.

20. produce pharmaceutical preparation in order to carry the method for bioactive medical compounds or genetic material for one kind, this method comprises: according to any synthetic method synthetic polymer among the claim 15-18 with bioactive medical compounds of this polymeric encapsulate or genetic material.

21. according to any one dendritic polymer or asymmetrical hyperbranched polymer of claim 1-8, wherein, described polymeric encapsulate bioactive medical compounds or is being carried the genetic material that is used for the treatment of.

22. according to any one dendritic polymer or the asymmetrical hyperbranched polymer purposes that is used to make pharmaceutical dosage form of claim 1-14, wherein, described polymeric encapsulate biologically active drug compound or carry the genetic material that is used for the treatment of.

23. according to any one dendritic polymer or the purposes of asymmetrical hyperbranched polymer in making the medicine of treatment and the biologically active drug compound of sealing or the genetic material same disease or the patient's condition of claim 1-14, wherein, this compound of described polymeric encapsulate or carrying gene material.

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