CC BY
12UDC 615.32:577.13.3
M. A. Vinter, I. S. Kazlouski, A. I. Zinchenko
PRODUCTION OF CHITOSAN NANOPARTICLES CONTAINING 3',5'-CYCLIC DIADENOSINE MONOPHOSPHATE
State Scientific Institution "Institute of Microbiology of the National Academy of Sciences of Belarus" 2 Kuprevich St., 220141 Minsk, the Republic of Belarus e-mail: [email protected]
The possibility to use natural biopolymer chitosan for the production of nanoparticles containing molecules of pharmaceutical^ valuable 3',5'-cyclic diadenosine monophosphate (cyclic di-AMP) was demonstrated. Experimental conditions were optimized to produce chitosan complexes with cyclic di-AMP (sized about 100 nm), which capacity in regard to cyclic di-AMP reached 38-40%. It was established that cyclic di-AMP is released from its complex with chitosan in a pH-dependent mode (more actively at pH 4.5 than at pH 7.5). The obtained results show that the studied nanocomplexes can be used for the pH-controlled delivery of cyclic di-AMP into cells.
Keywords: 3',5'-cyclic diadenosine monophosphate, nanoparticle, chitosan, adjuvant, chitosan dinucleotide complex, controlled delivery of drug to target cells.
Introduction
The apprehension of emerging new devastating pandemic outbreaks dictates the need to seek effective antiviral formulas [1].
Earlier, the authors [2] have proposed to apply pharmacologically attractive compound cyclic 3',5'-diadenylate (cyclic di-AMP) (Fig. 1), recently discovered in bacteria and archea [3, 4], as a universal antiviral agent playing a role of the inducer of species-specific endogenic interferon in nature [5, 6], which protects the body of vertebrata from various infectious pathogens.
However, this negatively charged natural dinucleotide remains unstable in blood vessels and can hardly penetrate into immunocytes [7].
This fact requires that scientists develop new
methods enabling the efficient delivery of cyclic di-AMP into pathological tissues and immune target cells.
In this regard our interest was focused on relatively safe natural polymer chitosan — the product of the partial deacetylation of chitin isolated from the shells of crustaceous species, mollusks, insects and fungi [8, 9].
Cationic characteristics of chitosan make the derived nanoparticles (NPs) an attractive material for the elaboration of novel systems providing the delivery of negatively charged drug substances to the target cell tissues [10].
Moreover, chitosan NPs combine the features and benefits of both chitosan and nanocorpuscules [11], capable per se of selectively accumulating
Fig. 1. Structural formula of cyclic di-AMP
within lymph nodes and stimulating the presentation of antigen and congenital immune responses not mediated by adjuvants [12].
To date, numerous techniques were developed for chitosan NPs production [13], yet the most popular methods are ionotropic gelation and polyelectrolyte complexing [14].
To produce chitosan complexes incorporating cyclic di-AMP, in this study we resorted to the method of ionotropic gelation because it is relatively simple and does not require toxic ligating agents and organic solvents.
Materials and methods
For the synthesis of its complexes with cyclic di-AMP, chitosan was provided by the company Acros Organics (Belgium). The stock material with the molecular weight of 100-300 kDa and the 75% deacetylation degree was dissolved in the 1% acetic acid to 0.1% concentration.
The aqueous solution (0.1%) of cyclic di-AMP, synthesized as described previously [15], was added dropwise to the above-mentioned compound at ambient temperature and continuous agitation with magnetic stirrer. The resulting suspension was centrifuged for 10 min at 10,000 g, and the sediment was washed twice with distilled water and subsequently was desiccated in the air at 60 °C during 24 h.
The efficiency of cyclic di-AMP integration into chitosan complexes was estimated via the spectrophotometric (X = 259 nm) determination of its concentration in the supernatant upon precipitation of complex particles. The parameter was calculated in accordance with the following formula 1:
a=m2 x ^ (1)
where A — incorporation efficiency (%),
m1 — the mass of cyclic di-AMP integrated into the particles,
m2 — the mass of cyclic di-AMP engaged in the reaction of complexing.
The capacity of chitosan complexes relative to cyclic di-AMP was evaluated by the formula 2:
E = m1 x 100, m3
(2)
where E — the capacity of complex prepara-
tion
ml — the mass of cyclic di-AMP integrated into the nanocomplex,
m3 — the mass of a dried complex.
The size of NPs was measured in collaboration with colleagues from the Institute of Bioorganic Chemistry, NAS of Belarus, by the method of dynamic light scattering using Zetasizer Nano (Malvern Instruments, UK) in compliance with the manufacturer's guidelines.
To release cyclic di-AMP from chitosan complexes, a sample of the complex was resuspended in distilled water or in a citrate-phosphate buffer. The suspension was incubated at room temperature, and aliquots were collected at defined time intervals. The elution rate was assessed via the spectrophotometric (X = 259 nm) determination of its concentration in the supernatant upon the sedimentation of complex particles.
The antibacterial activity of chitosan complexes with cyclic di-AMP was analyzed using the method described in the study [16]. The test cultures Escherichia coli BIM B-1033 and Staphylococcus aureus BIM B-1841 were provided by the Belarussian collection of nonpathogenic microorganisms, the Institute of Microbiology, NAS of Belarus.
Experimental data obtained in this research represent the confidence range of arithmetic means for 95% probability level.
Results and discussion
The initial investigation stage was devoted to the synthesis of NPs from chitosan and cyclic di-AMP. Mixing of chitosan and cyclic di-AMP solutions, according to the procedure described in the section "Materials and methods", resulted in the transparent, slightly opalescent solution. The component loading efficiency, NPs capacity with regard to the molecules of a ligand (cyclic di-AMP), and their size were chosen as the main parameters characterizing produced NPs. It was presumed that shifting of chitosan — the cyclic di-AMP ratio toward the latter component — is likely to promote the efficient binding of a ligand to the support and holding capacity of resultant NPs with respect to a cyclic di-AMP constituent.
Results of the experiment illustrated by Fig. 2 testify that with an increase in chitosan
(the cyclic di-AMP mass ratio above 1:3) the component loading efficiency tends to decline, whereas capacity of NPs in relation to immobilized cyclic di-AMP expands.
Experimental results evaluating the size of dinucleotide-chitosan complexes are reflected in Fig 3. It should be noted that size measurements were conducted for the complexes displaying, in
our opinion, the highest parameters of loading efficiency and elevated holding capacity.
It may be seen in Fig. 3 that all derived complexes are distinguished by nanosize dimensions. Their size tends to rise with an increase in the chitosan/ cyclic di-AMP mass ratio toward the latter component. Thus, we originally demonstrated the first evidence of chitosan binding with cyclic di-
Chitosan: cyclic di-AMP mass ratio Fig. 2. Binding of cyclic di-AMP to chitosan
Fig. 3. The size of chitosan/cyclic di-AMP NPs: empty chitosan NPs with TPP in the ratio 1:2 (A); chitosan NPs with
cyclic di-AMP in the mass ratio 1:1 (B), 1:2 (C) and 1:3 (D)
AMP and the production of nanosize complexes.
Noteworthy is that the produced chitosan/cyclic di-AMP complexes possess antibacterial activity, as stated in previous reports by other authors for chitosan NPs [17].
Fig. 4 illustrates the antibacterial activity of NPs resulting from the interaction of chitosan and cyclic di-AMP in 1:1 ratio during the experiments with two test cultures. It follows from the results presented in Fig. 5
Fig. 4. Antibacterial activity of chitosan NPs tested on the cultures E. coli BIM B-1033 (A) and St. aureus BIM B-1841 (B)
that the produced NPs show antibacterial activity towards both microbial strains studied.
To elucidate the prospects of applying chitosan/ cyclic di-AMP complexes for the delivery of an active compound to target cells, we staged an experiment releasing a dinucleotide from NPs under different conditions.
Fig. 5 provides the curves reflecting the extent of cyclic di-AMP liberation from the chitosan complex with time of incubation in distilled water and the citrate-phosphate buffer at various pH values (7.4, 4.5 and 1.5). Such pH values were motivated by normal blood acidity around 7.4 [18], while pH within lysosomes fluctuates in the
Elution rate, % 100
90
80
70
SO
50
range from 4 to 5 [19].
The citrate-phosphate buffer was selected to maintain pH of the solution in the optimal testing area.
It is evident from Fig. 5 that cyclic di-AMP was not eluted from nano-complex into water. In contrast, elution into the citrate-phosphate buffer (pH 7.4.) reached 38% by nearly 24 h. Under acidosis conditions (pH 4.5 and 1.5), the release of cyclic di-AMP from chitosan complexes upon 21 h attained 49% and over 90%, respectively.
The proven fact of cyclic di-AMP liberation from chitosan complexes evidences in favor of engaging the system studied into the prolonged
40 30
20
10
pH 1 5
-------
pH 4,5
___ --
__— pH 7,4
j
1 Water *
10
15
20 25
Time, h
Fig. 5. The pH-dependent release of cyclic di-AMP from complexes with chitosan
delivery of an active dinucleotide to target cells.
Conclusion
Earlier, we proposed using the inducer of endogenic interferon cyclic di-AMP as a universal antiviral agent. The advantage of this compound over other numerous interferon inducers is that cyclic di-AMP represents an element of a naturally evolved mechanism ensuring the universal protection of vertebrata from the attack of multiple viral pathogens. Unfortunately, a molecule of this dinucleotide has two negative charges presumably complicating its penetration into negatively charged virus-infected and malignant cells.
In the literature known, the process of the immobilization of active compounds on positively charged supports, including chitosan carriers, was described as a solution to the problem.
The present study showed the effects of the prolonged cyclic di-AMP release from chitosan nanoparticles in mildly acidic media and indicated the principal possibility of using the nanocomplexes studied for the pH-controlled delivery of cyclic di-AMP to target cells.
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М. А. Винтер, И. С. Казловский, А. И. Зинченко
ПОЛУЧЕНИЕ НАНОЧАСТИЦ ХИТОЗАНА, ВКЛЮЧАЮЩИХ 3',5'-ЦИКЛИЧЕСКИЙ ДИАДЕНОЗИНМОНОФОСФАТ
Государственное научное учреждение «Институт микробиологии Национальной академии наук Беларуси» Республика Беларусь, 220141, г. Минск, ул. Купревича, 2 e-mail: [email protected]
Показана возможность использования природного биополимера хитозана для получения наночастиц, включающих молекулы фармацевтически важного 3',5'-циклического диаденозинмонофосфата (цикло-ди-АМФ). Подобраны экспериментальные условия для получения комплексов хитозана с цикло-ди-АМФ (размером порядка 100 нм), емкость которых в отношении цикло-ди-АМФ достигает 38-40 мас.%. Установлен факт высвобождения цикло-ди-АМФ из его комплекса с хитозаном рН-зависимым способом (при рН среды 4,5 более активно, чем при рН 7,4). Полученные результаты свидетельствуют в пользу возможности применения изученных на-нокомплексов для рН-контролируемой доставки цикло-ди-АМФ в клетки-мишени.
Ключевые слова: 3',5'-циклический диаденозинмонофосфат, наночастица, хитозан, адьювант, хитозан-ди-нуклеотидный комплекс, контролируемая доставка лекарства в клетки-мишени.
Дата поступления в редакцию: 11 июля 2023 г.
