RU2599488C1 - Use of indocyanine green as a marker of nanoparticles - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and is intended for use of indocyanine green as nanoparticles optical mark containing medicinal substance of protein nature. Indocyanine is used as a nanoparticles matrix marker based on polylactic acid or copolymer of polylactic and glycolic acid with average size of not more than 500 nm, containing substance of protein nature. Using the invention enables to study internalization of nanoparticles (IN) and encapsulated active substance in parallel, without impacting on the optical properties of each mark - marked protein or peptide and marked IN with cells, their intracellular distribution.

EFFECT: marking of IN allows to study the pharmacokinetic parameters of IN, their bio-distribution and their safety in parallel with the active substance, that is important for introduction and wider application of IN in vivo.

1 cl, 6 tbl, 12 dwg, 5 ex

Description

The invention relates to biotechnology, in particular to the use of green indocyanin (ICZ) as an optical label for nanoparticles containing a protein substance of a medicinal substance.

Today, as a result of the development of the sphere of biotechnology at the commercial level, a huge number of peptide and protein preparations are produced. In the last decade, there has been an increase in interest in the creation and production of biological products intended for the treatment of a number of diseases with a significant unrealized need for medicine. Clinical development of drugs of these types is impossible without special technical equipment, in contrast to the traditionally used drugs based on small molecules. Oral administration of drugs is the most common method of administration, but it is usually impossible in the case of peptide and protein preparations. The main reason for the low bioavailability of biological products when taken orally is the pre-systemic enzymatic cleavage and the difficult penetration into the bloodstream. In recent decades, the mechanism of absorption of macromolecular preparations from the gastrointestinal tract (GIT), as well as barriers to absorption in the digestive tract, have been thoroughly studied. Various methods have been tested to overcome these barriers and strategies to develop safe and effective systems for oral protein ingestion. Oral administration of peptides and proteins still causes significant difficulties and is the subject of many pharmacological studies.

The transfer of protein-based drugs is hindered by the following:

- Most therapeutic peptides and proteins are hydrophilic and have a LogP value of <0. Thus, they cannot undergo transcellular absorption by passive diffusion.

- The sizes of the paracellular space are in the range of 10 and 30-50A °, and the paracellular path is impossible for the absorption of macromolecules, since it is limited by the relatively small hydrophilic molecules that can pass through such a space.

While some proteins are actively absorbed through the cell apical membrane of the cells of the small intestine, after binding to cell receptors, only a small fraction goes to the basolateral membrane and is released into the bloodstream in an unchanged form. Despite this, there is increasing evidence that a significant amount of peptides and proteins (sufficient for a pharmacological effect) can be absorbed, provided that they are protected from proteolytic enzymes in the digestive tract.

To increase the bioavailability of biological drugs when taken orally, a strategy aimed at increasing their penetration or using protease inhibitors as additives can be effective (see Innovations in the Delivery of Peptides. Business Insights, (2012), 1-242., A. Sood , et. al. Chem. Rev. 101, (2001), 3275-3303., Leone-Bay, M. Sato, et. al., Pharmaceut. Res. 18 (2001) 964-970., A. LeoneBay, KH Ho, et. Al., J. Med. Chem. 39, (1996), 2571-2578, S. Lee, et. Al., Diabetologia 48, (2005), 405-411., M. Kidron, et . al., Diabet. Med. 21, (2004), 354-357., SJ Wu, JR Robinson, J. Control. Release 62, (1999), 171-177.)

Although these methods can be successfully applied in the laboratory, they are still not recognized by most medical practitioners and regulatory authorities. The use of enzyme inhibitors during long-term therapy remains a controversial issue due to the possible absorption of unwanted proteins, impaired absorption of nutrient proteins and stimulation of protease secretion as a result of reverse regulation.

A strategy for modulating intestinal cell permeability through tight junctions (Tight Junctions) was studied to stimulate paracellular transport of drug molecules. The ZOT toxin (Zonula Occludens toxin), chitosan, thiolated polymers and the Pz peptide show a pronounced ability to increase the absorption of macromolecular preparations. However, it is likely that this strategy may also raise security concerns. After opening close contact, the transfer of not only drugs, but also potentially toxic or unwanted molecules present in the digestive tract is stimulated.

Therefore, in this case, a suitable option may be the use of drug delivery systems (ADDS), such as hydrogels, nanoparticles, microspheres, and lipid-based drug delivery systems (e.g., nanoemulsions, liposomes, and solid lipid nanoparticles), which are aimed at protecting peptides and proteins from enzymatic degradation and which are able to modulate drug transfer.

In particular, when working with biodegradable substances, SDLV at the nanoscale (<1 μM) has the following advantages:

- suitable for drug delivery with hydrophobic, amphipathic and hydrophilic properties;

- well biocompatible due to biodegradability and low toxicity;

- can serve as a means of controlled release of a drug substance in the body;

- may be administered in various ways, including oral.

Abbreviations Used

SDLV - drug delivery system LF nanoparticles

PMHC - a copolymer of poly (lactic-co-glycolic acid)

PMK - poly (lactic acid)

BSA - Bovine Serum Albumin

FITS - fluoresceinisothiocyanate

IC3 - Indocyanine green

SEM - scanning electron microscope

MTS - 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium 3- (4,5-dimethylthiazol-2-yl) -5 - (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium

Papp - coefficient of visible permeability.

DMF - dimethylformamide

DXM - Dichloromethane

TFA - trifluoroacetic acid

PVA - polyvinyl alcohol

DDV - bidistillate of water

SDS - sodium dodecyl sulfate

EI - Encapsulation Efficiency

K3 - load factor

SP - the content of the drug

IPD - polydispersity index

IKS - artificial intestinal environment

VP - drug release

PMS - Phenazine Metasulfate

KLSM - Confocal laser scanning microscopy.

Scheme 1 shows a labeled peptide.

Fig. 1 HPLC chromatogram of the crude peptide-FITC 10-mer, performed by an Alliance Waters chromatograph using a 2,475 multi-wavelength fluorescence detector at a wavelength of 485/528 nm and an MS analysis of time-of-flight laser desorption ionization using a matrix.

Fig. 2 HPLC chromatogram of the purified product. The purity of the peptides and Mw was determined using a Voyager DE-Pro MALDI-TOF (ABI) instrument (B) using α-cyano 4-hydroxycinnamic acid in 0.1% THC: ACN (1: 1) as a matrix in linear-positive mode .

Fig. 3 SEM of a suspension of nanoparticles containing BSA-FITZ prepared from a polymer ΠΜΓΚ-RG 503 using MeCl ₂ as a solvent. Particles were created by the double emulsion method using 0.5% sucrose as a cryo- and lyoprotectant. A and B show LF # 9 (table 1A), and C and D show LF # 31-F, labeled ICZ (table 1A).

Fig. 4 SEM of a suspension of nanoparticles containing FITC peptide prepared from ΠΜΓΠΜ-RG 503 polymer using MeCl ₂ as a solvent. Particles were created by the double emulsion method using 5% sucrose as a cryo- and lyoprotectant. A and B show LF # 28 (table 1B), and C and D show LF # 47, labeled with ICZ (table 1B).

Fig. 5 SEM of lyophilized powder of nanoparticles No. 48 containing FITC peptide (table 1B)

Fig. 6 SEM of a suspension of nanoparticles containing FITC peptide prepared from a ΠΜΓΠΜ-RG 503 polymer using MeCl ₂ as a solvent. Particles were created by co-dissolving the m / v emulsion using 5% sucrose as a cryo- and lyoprotector. A and B show LF # 46 (table 1C), and C and D show LF # 55, labeled ICZ (table 1C).

Fig. 7 SEM of a suspension of nanoparticles No. 59 (Table 1C) containing an FITC peptide prepared from a ΠΜΓΚ-RG 503 polymer using MeCl ₂ as a solvent. Particles were created by co-dissolving the m / v emulsion using 5% sucrose as a cryo- and lyoprotectant using ultrasound treatment.

Fig. 8 SEM of a suspension of nanoparticles No. 56, 57, 58 labeled with ICZ (Table 1B) containing an FITC peptide prepared from ΠΜΓΚ-RG 503 (A) polymer, Lactel A 9 (B) or Lactel All (C) using MeCl ₂ in as a solvent. Particles were created by the method of a double emulsion in / m / in using 5% sucrose as a cryo - and lyoprotector.

Fig. 9 SEM of a suspension of nanoparticles No. 54, 60, 61 labeled with ICZ (Table 1C) containing an FITC peptide prepared from ΠΜΓΚ-RG 503 (A) polymer, Lactel A 9 (B) or Lactel All (C) using EtAc as solvent. Particles were created by co-dissolving m / v using 5% sucrose as a cryo- and lyoprotector.

Fig. 10 Kinetics of the cumulative release of FITC peptide from nanoparticle formulations prepared by the double emulsion method (A) and the co-dissolution method of m / v (B) in ICS.

Fig. 11 The effect of NP-based formulations containing BSA-FITC on the viability of Caco-2 cells, evaluated by the MTS method.

Fig. 12 Distribution of SACO-2 FITC peptide in cells (green) after 4 hours of incubation with various formulations. Treated Caco-2 cells were examined using a Fluoview 300 confocal laser scanning microscope (KLSM, Olympus, PA, USA).

The most well-studied class of polymers, from the point of view of toxicological and clinical data, are aliphatic poly (ether) s, consisting of lactic and glycolic acid. Their respective polymers, poly (lactic acid), poly (glycolic acid) and poly (lactic-co-glycolic acid) copolymer have found wide commercial application as nanoscale drug delivery systems. Poly (lactide-co-glycolide) (PLGA) is the "gold standard" of biodegradable polymers. The properties of these copolymers can be modified by changing the ratio of PLA and PLG, which is reflected in an increase in the biodegradation time from several days to several weeks with an increase in the content of PLA in SDLS.

Generally speaking, nanoscale stimulates the transfer of particles through the epithelium of the mucous membranes. It was shown that 100 nm particles of poly (lactic-co-glycolic acid) (PMHC) were distributed throughout the submucosal layer, while 10 mm particles were mainly localized on the superepithelial layer of tissues. In their totality, nanosized carriers consisting of biocompatible polymers are considered promising for the development of an oral delivery system for macromolecules.

PMHC and PMA are highly biocompatible and biodegradable. They have been used since the eighties for various purposes in vivo (for biodegradable implants, controlled release of drugs). Since the absorption of particles by intestinal cells depends on the size of the particles, their size is one of the most important conditions for successful oral delivery of nanoparticles. In our study, PMHC was chosen as the most suitable carrier of drugs; in this case, various types of PMHC were used in the creation of nanoparticles, as well as polylactic acid itself, as the extreme type of copolymer.

As a result of the studies, the ADDS in the form of NPs labeled with an optical tag with the active substance of a protein nature were developed.

The approximate quantitative composition of ADDS is shown in table 2.

Composition notes:

1) The polymer is the main component of the nanoparticles, the emulsifier can be practically removed in the technical process, the lyoprotector can not be used, and the peptide is effective in microdosages, it may turn out that the bulk will fall on the polymer.

2) For polymer-based nanoparticles, up to about 60% loading is possible.

3) The emulsifier can be either practically removed in the process, or act not only as an emulsifier, but also as a lyoprotector.

4) The invention can be implemented in the form of a suspension of nanoparticles (lyoprotector - 0%), and in the form of a solid dosage form obtained by lyophilization, then a lyoprotector (for example, mannitol) can be used as a filler.

Effective for delivery of the active substance are nanoparticles,

having an average size of not more than 500 nm. From well-known studies by Anne des Rieux, et. al., Transport of nanoparticles across an in vitro model of the human intestinal follicle associated epithelium, European journal of pharmaceutical sciences 30 (2007) 380-391 and G.J. Russell-Jones, et. al., Lectin-mediated transport of nanoparticles across Caco-2 and OK cells, Int. J. Pharm. 190 (1999) 165-174, in which the transport of nanoparticles with a maximum size of 500 nm was studied, it subjectively follows that this is the upper boundary of particles with significant penetration (RU 2145498).

An important objective of this study was the development of nanoparticle-based delivery systems that can effectively track (in vitro and in vivo) both the carrier and the active substance. Fluorescence microscopy techniques are becoming important tools in preclinical studies. One of the main reasons for developing new fluorescent probes for use in the clinical setting is the need for near-infrared tracers that provide a high signal-to-background ratio. The spectral properties of these fluids thus define a narrow “optical window”, between 650 and 900 nm, in which light absorption by the main tissue components, such as hydroxy and deoxy hemoglobin (λ _max <600 nm) and water (λ _max > 1150 nm) is minimal, therefore, it allows light to penetrate much deeper, as a rule, to a depth of several centimeters. In addition, tissue auto-fluorescence also decreases in the near infrared region compared to the visible range. Consequently, the use of the near-infrared dye can circumvent the disadvantages of classical imaging methods and opens up new analytical possibilities (Altinoglu EI, Adair JH. Near infrared imaging with nanoparticles, Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2010; 2 (5): 461-77). Indocyanine green (ICG, emission ≈800 nm), the only fluorescent dye of the near infrared region with an absorbing / emitting wavelength> 700 nm, which has now been approved by the Food and Drug Administration for human use.

An important criterion when choosing an optical label is the following parameters - low toxicity and low immunogenicity. Unfortunately, many near-infrared optical labels have the following disadvantages: discoloration, non-specific binding to proteins, biological and chemical degradation, and their aggregation, which significantly weaken the ability to diagnose the label signal.

These deficiencies in optical labels have made rational the development of alternative optical labels such as labeled nanoparticles (Altinoglu EI, Adair JH. Near infrared imaging with nanoparticles, Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2010; 2 (5): 461-77). Unlike low molecular weight organic dyes, the use of fluorescent nanoprobes can increase the resolution of the system - reduce the threshold of sensitivity (the best optical properties in vivo) and study the biodistribution of delivery systems by determining the fluorescent signal. Semiconducting nanocrystals (Quantum dots, QD), gold nanoparticles (gold NP) and biodegradable nanoparticles are the most optimal optical labels, among them biodegradable nanoparticles are most suitable for their in vivo use compared to semiconducting nanocrystals and gold nanoparticles due to their greater safety.

For this reason, we have found it highly advisable to develop nanoparticles based on biodegradable polymers labeled with ICZ. Indocyanine is an amphiphilic dye. Therefore, it can be encapsulated in the hydrophilic space of the LF, as described in the article Subhash NM. (Subhash HM. Et. Al., Optical detection of indocyanine green encapsulated biocompatible poly (lactic-co-glycolic) acid nanoparticles with photothermal optical coherence tomography, Opt Lett. 2012 Mar 1; 37 (5): 981-3). This reference shows that indocyanine can be encapsulated in the inner hydrophilic space of the NP. This approach allows one to increase the stability of the optical tag, but it makes it difficult to study the pharmacokinetic parameters of the NP, their bio-distribution, and their safety, since any violation of the integrity of the NP membrane or their dilution, usually occurring in the gastrointestinal tract, will cause the optical tag to exit the hydrophilic space of the NP hydrophilic outer space. Moreover, the encapsulation of the optical label and the active substance in the same space will adversely affect the loading of the optical label and especially the active substance, in this case the protein. In our case, we propose to include the ICZ in the bass for marking the bass itself. This approach allows tracking the internalization of NPs and the encapsulated active substance in parallel, without affecting the optical properties of each label, in our case, the labeled protein or peptide with cells, and their intracellular distribution. In our case, labeling of the NPs themselves allows us to further study the pharmacokinetic parameters of NPs, their bio-distribution and their safety in parallel with the active substance, labeled protein or peptide. These properties are a very important characteristic for regulatory bodies, since to date the toxicity of NPs has not been fully studied and this is an obstacle to wider introduction and wider use of NPs in vivo.

We selected two molecules as models of a drug substance of protein nature — BSA-FITC and decapeptide-FITC glowing in the visible spectrum — which allowed us to efficiently characterize the obtained nanoparticles by parameters including morphology, size, content of the preparation (SP), and kinetics of drug release in simulated intestinal fluid, intracellular distribution and transport in the intestine.

The possibility of carrying out the invention is confirmed, but not limited to examples.

Example 1 Preparation of Labeled Model Proteins

Materials

BSA-FITZ, ammonium molybdate, 4-amino-2-naphthyl-4-sulforeagent, green indocyanine, PMGC-poly (lactic-co-glycolic acid) (50:50) RG 503, polyvinyl alcohol, pluronic® F-127 were purchased from Sigma-Eldrich (St. Louis, Missouri, USA).

1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- (lissamine rhodamine B sulfonyl) (ammonium salt), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol were purchased from Avanti Polar Lipids (Alabaster, Alabama, USA).

Peptide Properties

The model decapeptide was synthesized by solid-phase peptide synthesis (TFSP) by Sigma-Eldrich (WYPA10, Rehovot, Israel).

Peptide sequence

FMOC-Trp-Tyr-Phe-Ser-Tyr-Tyr-Leu-Lys-Prol-Ala-MBHA-resin,

where the side chains of Trp, tyr, Ser and Lys are protected

Peptide labeling

For this, the resin-bound peptide was first increased in DMF for 60 minutes, and 9-fluorenylmethoxycarbonyl was removed by incubation in 20% piperidine in DMF for 20 minutes and then another 10 minutes, followed by washing in DMF (× 4) and DXM (× 4). After that, 4 eq of the binder Fmoc-6-Ahx-COOH (HSX-Fmoc) was coupled to the peptide (30 min incubation) after preliminary activation of 1H-7-azabenzotriazol-1-yl) -1,1,3,3- tetramethyl with uronium hexafluorophosphate (4 eq, HATU 2), hydroxybenzotriazole (4 eq, HOBT) and N, N-diisopropylethylamine (4 eq, diisopropylethylamine) for 2 min. Then, the 9-fluorenylmethoxycarbonyl binder was removed as previously described.

Then, 4 equivalents of fluoresceinisothiocyanate (FITC) were bound to the peptide using a binder in the presence of diisopropylethylamine by incubation for 24 hours, followed by washing with DMF (× 4) and DCM (× 4).

The peptide and its side protecting groups were removed from the resin by incubation for 2 hours using trifluoroacetic acid / distilled deionized water / phenol / triethylsilane (88/5/5/2 v / v). The labeled peptide (Scheme 1) was precipitated with cold diethyl ether and its excess was removed with argon. The procedure was repeated (Fig. 1).

The structural formula is presented in scheme 1

C ₉₇ H ₁₁₁ N ₁₅ O ₂₀ S

Exact weight: 1837.79

Molecular mass: 1839.07

Mg / equiv: 1838.79 (100.0%), 1837.79 (93.5%), 1839.79 (63.1%), 1840.80 (18.5%), 1840.79 (7.7%), 1841.80 (7.0%), 1838.78 (5.9%), 1840.78 (4.9% ), 1839.78 (4.4%), 1841.79 (4.1%), 1842.80 (2.1%), 1842.79 (1.2%)

C, 63.35; H, 6.08; N, 11.42; 0.17.40; S, 1.74 Purification of the peptide by HPLC (Fig. 2):

- Column: Agilent Luna C18-2 250 × 4.6

- Column temperature: 40 ° C

- Volume of injection: 100 μl

- Solution A: 0.1% trifluoroacetic acid (TFA); Solution B: acetonitrile

- Gradient (tab. 3)

Example 2 Preparation of PMGC-based Nanoparticles (NPs) with Protein Model

PMHC RG 503 (50:50) and Lactel PMHC were used as material for nanoparticles, methylene chloride (MeCl ₂ ) or ethyl acetate (EtAc) were used as organic solvents, polyvinyl alcohol (PVA) or Pluronic F-127 (PF-127 ) were used as emulsion stabilizers, and the FITC-BSA or FITC 10-mer peptide were model proteins used to encapsulate NPs. Indocyanin Green (ICG) was used as a marker for LF.

The nano-encapsulation procedure was based on the double emulsion (v / m / v) and m / v co-dissolution methods. In the double emulsion method, PMHC is dissolved in an organic solvent, and then FITZ-BSA is dissolved in DDV containing 2% PVA, and FITZ-peptide is dissolved in methanol: DDV (1: 4 w / w) containing 1% BSA and 2% PVA add to the solution of PMSA at various ratios of PMSA / preparation and homogenize at 11,000 or 15,000 rpm for 2 minutes, or ultrasound in an ice bath for 1 min. This v / m emulsion is then poured into an emulsifying solution in DDV at various phase ratios and again homogenized at 11,000 or 15,000 rpm for 2 minutes or ultrasound in an ice bath for 1 minute.

Using the co-dissolution method, m / in PMHC is dissolved in an organic solvent, and then FITC - the peptide dissolved in methanol is added to the PMHC solution at different proportions of PMHC / protein preparation and homogenized at 15,000 rpm for 2 min or by ultrasound in an ice bath within 1 min. This v / m emulsion is then poured into an emulsifying solution in DDV at various phase ratios and again homogenized at 11,000 or 15,000 rpm for 2 minutes or ultrasound in an ice bath for 1 minute.

In both cases, the emulsion was stirred for 1-2 hours to remove the organic solvent, and then the residual solvent was evaporated on a vacuum evaporator (Labconco, Missouri, USA). The prepared suspensions were then washed 3 times with a reduced deuterium content in a centrifuge at 14,000 rpm for 25 minutes to eliminate the non-encapsulated preparation. Solids were collected and immersed in liquid nitrogen and then lyophilized (Labconco, Missouri, USA) overnight using 5% sucrose as a lyoprotector. The number of produced NPs ranged between 37-81% (Tables 1A and B).

Example 3 Assessment of the physicochemical characteristics of low frequencies

Preparation of samples for analysis by scanning electron microscope:

For this, PMHC-based nanoparticles were suspended in DDV to obtain a 0.25-1% suspension and sonicated for 60 seconds. One drop of the suspension was placed on a silicon substrate, dried and coated with gold. In another preparation, the dried NPs were immobilized, placed on a silicon substrate, and also coated with gold. After that, in both cases, PMGC-based nanoparticles were studied under a MagellanTM 400L super-high resolution scanning electron microscope (IPPE, Oregon, USA).

Determination of size and zeta potential

The size distribution and zeta potential of protein-containing PMGC-based nanoparticles were measured at 25 ° C using a Zetasizer Nano-Z instrument, Malvern Instrument Ltd., Malvern, UK. For this, 1 mg of protein-loaded PMHC-based nanoparticles was diluted in 1 ml of filtered DDV and examined.

Evaluation of the effectiveness of encapsulation of the drug in NP based on PMHC

To do this, 2 mg of NP were dissolved in 1 ml of 0.1 M NaOH / 0.5% SDS by shaking. 250 μl of the dispersions were immediately checked for fluorescence on an Odyssey scanner (LI-COR Biosines, Lincoln, Nebraska, USA) at sensitivity 2 and emission of 800 on a 96-well plate, and then incubated at 37 ° C for 2 hours . After that, the samples were centrifuged for 5 min at 14000 rpm to obtain a clear supernatant. 250 μl of the supernatant was collected and analyzed for fluorescence using a multi-mode tablet reader (BioTech, Vermont, USA) at ex485 / em525 with a sensitivity of 50.

The encapsulation efficiency of the drug (EI) was evaluated using a standard drug curve of 1% 0.1 M NaOH / 0.5% SDS after 2 hours of incubation at 37 ° C in accordance with the following equation:

EI (%) = 100 × X / Y,

Where:

X is the amount of model peptide in the obtained sample, mg;

Y is the amount of model peptide introduced in those. process, mg;

Additionally, the drug content in the LF was measured gravimetrically in accordance with the ratio of the drug / LF in / in × 100.

results

Comparative characteristic of PMHC-based NPs containing FITC-BSA or FITC peptide prepared by the i / m / v emulsion method.

The inclusion of ICG greatly influenced the effectiveness of low frequencies: short circuit, EI and SP fell sharply (table 1A), while their size increased sharply (table 1A, compare C and D with A and B in figure 3).

In the case of formulations containing FITC-peptide, as in the case of formulations with FITC-BSA, labeling of preparations with an increased amount of ICZ reduces the short circuit, EI, SP and increases the volume of formulations (compare LF No. 28, 43 with LF No. 40 (ratio drug / PMHC - 1/30) and 47 (drug / PMHC ratio - 1/10), respectively, in table 1B). Please note that an increase in volumes was not detected by DLS, but was detected by SEM (Figure 4C, D), and DLS does not detect large (micron) particles. Replacing the PMHC solution with EtAc improved the EI of the LF (compare LF # 40, 47 with LF # 56 in Table 1B) and the morphology analyzed by SEM (data not shown). The best composition in all respects was the investigated composition No. 48 (drug / PMHC ratio 1/10, table 1B and figure 4). The lyophilized composition No. 48 (dry form), shown in Figure 5, demonstrated that the nanoparticles formed were spherical nanoscale particles (100-200 nm). An additional selection parameter, which was observed to be exceptional for LF # 48, was the performance of the process; massive particle formation was achieved, and it is shown in Figure 4 and in Table 1B (obtained NPs). The use of Lactel PMHC polymers is inferior to Bhoeringer 503 PMHC, since in one case large NPs (LF No. 57, Table 1B) were formed of irregular shape, and in the other (LF No. 58, Table 1B) EI was quite low. Comparison of the LFs in Figure 8 using SEM obtained from PMGK RG-503 (LF No. 56, scale A - 100 μmol), Lactel 9 (LF No. 57, scale B - 20 μmol) or Lactel A11 (LF No. 58, scale C - 100 μmol), shows that the use of Lactel PMHC led to the formation of large NPs.

The use of a low amount of ICG (0.5 μg) was in all cases more preferable than a high amount of ICG (2.5 μg) (data not shown).

Again, the values of the polydispersity index (SDI) of NPs prepared in the study are consistent with the formation of a rather heterogeneous population (SDI> 0.3).

Comparative characteristic of PMHC-based NPs containing FITC peptide prepared by co-dissolving m / v emulsions.

The second method of co-dissolving the m / v emulsion was tested for the preparation of NPs containing a peptide because of the excellent solubility of the FITC peptide in an alcohol solvent such as methanol. When using this method, surprisingly, LFs without ICZ demonstrate lower performance (compare LF # 46 with LF # 53 and 55, Table 1C, Figure 6).

Adding the peptide first to alcohol and then to methylene chloride gave a clear solution. The peptide was probably solvated by hydrophilic hydrogen bonds forming alcohol clusters around the molecule, which could not be replaced by an excess of methylene chloride. When ethyl acetate was used as the polymer solvent, the preparation precipitated from the alcohol solution, resulting in a cloudy suspension. A more hydrophilic solvent, ethyl acetate, reacted with alcohol and desolvated a peptide that was practically insoluble in ethyl acetate. This effect does not affect the effectiveness of the LF (compare LF No. 51, 54 with 53, 55 in table 1C).

The main disadvantage of this method of preparing LFs was their large size (Figure 7). Therefore, to prepare the emulsion m / v, sonication was tested, rather than homogenization. This method made it possible to reduce the size of the formed LFs (LF No. 59 in Table 1C and Figure 7) and to achieve good EI and SP indicators.

A comparison using low-power SEM in Figure 9 obtained from PMGK RG-503 (LF No. 54, A), Lactel A9 (LF No. 60, B) or Lactel A11 (LF No. 61, C), shows that the use of Lactel PMHC led to the formation of large low frequencies. The preparation of NPs with a 4-fold amount of the drug (drug / PMHC ratio is 1 / 7.5 instead of 1/25 or 30 for all drugs) was successful in the case of a low (0.5 μg) amount of ICC, and we were able to get a high SP peptide in 5.6% (LF No. 63 in table 1C).

Example 4 Evaluation of LF Drug Release

To do this, 100 μl of 2 mg NPs were first dispersed in 250 μl of simulated intestinal fluid (IR, US Pharmacopoeia standard, pH 6.8) and checked for fluorescence using a multi-mode tablet reader (BioTech, Vermont, USA) at ex485 / em525 with a sensitivity of 70 immediately after preparation to determine the total fluorescence.

The NPs in the indicated amount were dispersed in 250 μl of IR and incubated in 250 μl of IR for 2, 4 and 24 hours at 37 ° C with shaking at 50 rpm. At the end of the incubation period, 500 μl of incubation medium was placed in Eppendorf tubes and scrolled at 14,000 rpm for 12 or 25 minutes, respectively. Further, this medium was analyzed for fluorescence using a multi-mode plate reader (BioTech, Vermont, USA) at ex485 / em525 with a sensitivity of 70 and returned to the incubation medium. The release of the drug (VP) was evaluated according to the standard drug curve in the ICS immediately after preparation and after incubation for 2, 4 and 24 hours at 37 ° C with shaking at 50 rpm at 37 ° C in accordance with the following the equation:

VP (%) = 100 × W / Z,

Where:

W is the amount of model peptide transferred to the incubation medium, mg;

Z is the quantitative content of the peptide in the sample taken for analysis, mg.

results

Kinetics of release of BSA-FITC and FITC peptide from NP to ICS

The PMHC copolymer undergoes degradation by hydrolysis or biodegradation by cleavage of its basic ether bonds into oligomers and, finally, monomers. The degradation of the PMHC copolymer is a collective process of volume diffusion, surface diffusion, volume erosion and surface erosion. A two-phase curve is usually observed for drug release as a result of biodegradation of PMHC. An “explosive” release of a drug depends on the type of drug, the concentration of the drug, and polymer hydrophobicity. The drug on the surface, in contact with the medium, is released depending on the solubility, as well as the penetration of water into the polymer matrix. In the second phase, the drug is released gradually through a thick layer with a small amount of the drug. “Explosive” release of protein preparations from NPs is also a problem, since it reduces the ability of a protein-based drug to achieve its goal due to degradation.

As can be seen in Figure 10, the “explosive” release of the FITC peptide was eliminated in all NPs prepared by both the i / m emulsion method (Figure 10A) and the m / w co-dissolution method (Figure 10 B) with Τ ½ more than 48 h

Example 5 Study of cell cultures.

Cell culture

The Saso-2 human colon carcinoma cell line was obtained from the American Type Culture Collection (ATCC, Manassas, Virginia). Saso-2 cells are grown in the complete minimum essential medium Eagle, modified by the method of Dulbecco (DMEM). Cell lines were stored at 37 ° C in a 5% CO ₂ water jacketed incubator.

Cell Proliferation Assay

First of all, the cytotoxicity of the prepared formulations was evaluated. The lack of cytotoxicity was assessed by MTS analysis using a CellTiter 96® AQ _{ueous kit} consisting of solutions of the new tetrazolium compound [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl ) -2H-tetrazolium, an internal salt, MTS] and an electronic coupling reagent (phenazine methasulfate) PMS, where MTS is bioreduced by cells to formazan product, which is soluble in the culture medium, and the absorption of formazan product can be measured at 490 nm. For this, Caco-2 cells were seeded in a 96-well plate at 4 × 10 ⁴ cells / ml (8 × 10 ³ / well) and allowed to adapt and grow for 48 hours. Then 50 μl of the studied compounds was added to the studied wells and incubated for 4 or 24 hours a day. At the end of the incubation period, the incubation medium was removed, the cells were washed with PBS and incubated in fresh medium for 24 hours, and then the cell viability was tested using the MTS assay.

Assessment of cell inclusion NP

Confocal laser scanning microscopy (CLSM) was used to evaluate the effectiveness of cell uptake. For the analysis of CLSM, Saso-2 cells were plated on films covered with a glass lid in 8-well plates at a density of 5 × 10 ⁴ cells / cm ² . After culturing for 48 hours, the culture medium was removed and the cells were washed twice using culture medium without phenol red dye. After that, 0.4 ml of the NP containing the preparation was added, diluted in a culture medium without phenol red dye in non-toxic concentrations, and the cells were incubated for 4 h at 37 ° C. At the end of the incubation period, the sample solutions were removed, the cells were washed three times in a culture medium without phenol red dye and studied using a Fluoview 300 confocal laser scanning microscope (Olympus, PA, USA).

results

The Saso-2 cell line, obtained from human colorectal cancer, became an in vitro model for determining the potential absorption of the drug through the walls of the human intestine. When cultivated on semipermeable membranes, Caco-2 cells differentiate in a highly functional epithelial barrier with pronounced morphological and biochemical properties similar to the epithelium of the small intestine.

Thus, the potential toxicity of NPs (Figure 11) was evaluated using the MTS method after 24 hours of incubation with Caco-2 cells. Figure I shows that all formulations were non-toxic at any of the concentrations studied.

Regarding the cellular distribution of low frequencies. LFs prepared by the i / m method were compared by their distribution in Caco-2 using labeled peptide (green staining) and ICZ (red staining) with an increase in the number of ICZ (LF # 51 and 53, respectively). Our results show that NPs labeled with a large number of ICGs (NP # 53) demonstrate both measurable efficacy (see Table 1B) and low cellular peptide uptake (Figure 12). According to the KLSM images, LF number 48 was chosen as the best composition.

Both compositions (NP 55 and 59), prepared by the co-dissolution method with IM, showed good cell distribution (inserts in Figure 12) and peptide delivery to the cells. Evaluated in all cases, the free FITC peptide showed a very low cell distribution (Figure 12).

Thus, the inclusion of ICH in NP has a huge impact on the characteristic properties of NPs, such as NP formation, NP size, EI of model peptides: FITC-peptide and FITC-BSA.

We have for the first time proven the fundamental possibility of using ICC as a label for NP based on PMHC and PMA with protein encapsulated medicinal substances to study the mechanism of penetration and distribution

NPs and the included substance in intestinal cells, followed by a study of the pharmacokinetic parameters and biodistribution of NPs and an assessment of their potential toxicity.

Claims (1)

The use of indocyanin as a marker matrix of nanoparticles based on polylactic acid or a copolymer of polylactic and glycolic acid with an average size of not more than 500 nm, containing a substance of protein nature.

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