rus
Issue #7-8/2024
A.I.Akhmetova, T.O.Sovetnikov, A.D.Terentev, I.V.Yaminsky
SCANNING CAPILLARY MICROSCOPY AS A TOOL FOR NANOCAPILLARY PRINTING
SCANNING CAPILLARY MICROSCOPY AS A TOOL FOR NANOCAPILLARY PRINTING
DOI: https://doi.org/10.22184/1993-8578.2024.17.7-8.418.425
Controlled manipulation of cultured cells and local delivery of macromolecules and substances are still unsolved problems in experimental biology. Intracellular injection of various therapeutic agents, including biologics and supramolecular agents, is difficult due to natural biological barriers required to protect the cell. Efficient delivery of nucleic acids, proteins, peptides and nanoparticles is critical for clinical implementation of new technologies that can benefit the treatment of diseases using gene and cell therapy. Using a capillary, it is possible to locally apply a desired substance to a cell or even introduce it into the cell, and then evaluate its effect on morphology using scanning capillary microscopy (SCM) tools. These capabilities make the capillary microscopy method promising for biomedical purposes.
Controlled manipulation of cultured cells and local delivery of macromolecules and substances are still unsolved problems in experimental biology. Intracellular injection of various therapeutic agents, including biologics and supramolecular agents, is difficult due to natural biological barriers required to protect the cell. Efficient delivery of nucleic acids, proteins, peptides and nanoparticles is critical for clinical implementation of new technologies that can benefit the treatment of diseases using gene and cell therapy. Using a capillary, it is possible to locally apply a desired substance to a cell or even introduce it into the cell, and then evaluate its effect on morphology using scanning capillary microscopy (SCM) tools. These capabilities make the capillary microscopy method promising for biomedical purposes.
Теги: living systems local substance delivery microdosing nanoparticles scanning capillary microscopy живые системы локальная доставка вещества микродозирование наночастицы сканирующая капиллярная микроскопия
INTRODUCTION
The cell is endowed with a dynamic mechanism, and access to it is strictly regulated by intracellular processes. Cells transmit information to each other through molecules, DNA encodes RNA and proteins, proteins transmit signals and act as building blocks of cellular structure, lipids form the membrane and store energy. Traditional methods of substance delivery can interfere with cellular function, limiting their applicability. Introducing molecules and substances into cells is an important experimental step in understanding its function, in controlling the cell and reprogramming its behaviour.
There are different ways to deliver substances into mammalian cells: by lipid vesicles and virus-like particles, by chemical transfection, electroporation and micro- and nano-injections.
Nanoinjection is an intracellular delivery process using nano-needles [1-3]. Nano-needles are generally not harmful to cells, including neurons, cardiomyocytes and immune cells [4-6]. Nanoinjection increases efficiency of plasmid DNA (pDNA) transfection in hard-to-transfect cells [7].
Not only nano-needles can be used for injection, but also glass capillaries with micron-sized exit hole diameters can be used to mechanically penetrate the plasma membrane, which also allows delivery of molecules. However, the large size of the capillary tip (typically 0.5 to 75 µm in outer diameter) relative to the cell size can deform the membrane during injection, disrupting the actin cytoskeleton and altering the cell morphology.
Using a puller (e.g., P2000, Sutter Instruments), capillaries with a minimum exit hole radius of up to 10 nm can be fabricated. Using an electrode placed in the capillary, the transport of molecules can be monitored by applying a suitable voltage and measuring the ionic current. The polarity and voltage value depend on the charge of the delivered molecule, and the voltage value should not exceed ±1 V [8, 9]. Moreover, the small size of the capillary tip relative to the cell allows not to deform the cell membrane. Translocation of one macromolecule through the capillary tip results in a detectable change in the measured ionic current, which can be used to characterise and quantify the number of molecules passing through the capillary [10, 11].
In [12], a platform is described that uses capillaries to deliver specific amounts of macromolecules into cultured cell lines and primary cells with resolution at the single molecule level. The capillary is used both as a probe of a scanning capillary microscope and as a needle for injection. The SCM is used to settle over the cell surface before the capillary is inserted to a predetermined depth into a specific location in the cell. With this setup, the quantitative delivery of DNA, globular proteins and protein fibrils into cells has been demonstrated with resolution at the single molecule level. And it is also demonstrated that delivery leads to a change in the phenotype of the cell.
The results on application of scanning capillary microscopy for local delivery of nanoparticles are also known [13, 14]. In this regard, the question of calculating their number that has passed through the holes of the nanocapillary arises. This approach will allow not only local but also high-precision delivery of nanoparticles to the cell surface.
EXPERIMENTAL RESEARCH
In order to develop the nanocapillary printing techniques, our group present a technique for counting the deposited particles number. The idea consists in selecting a capillary opening commensurate with the particles to be delivered (deposited), in which case the nanoparticle exit from the capillary (under the action of the applied potential difference) will be accompanied by the forming of a kind of barrier to the passage of ions, the value of the ionic current will decrease.
The known results [15-17] on the current behaviour description through the nanocapillary aperture show that over a wide range of end aperture size, the current through it can be described by Ohm’s law. The magnitude of the ionic current flowing through the aperture in the capillary is determined by the expression:
(1)
where – saturation current (away from the sample surface), z – sample surface distance, А – coefficient, taking into account the capillary geometry, determined through the relation [17]:
(2)
where – aperture outer diameter, – aperture inner diameter, L – distance from the capillary end to the electrode inside the capillary. The saturation current flowing through the nanocapillary at a large distance from the sample is mainly determined through the capillary resistance Rp via Ohm’s law:
(3)
where U – electrode voltage.
For resistance of the nanocapillary the following relation is valid:
(4)
where – conductivity of electrolyte medium, – inner radius of a capillary, – capillary apex angle (on average from 3° to 5°).
Using also the expressions for determining the size and resistance of the capillary (3, 4) and the current near the surface (1), and neglecting the constant components, it is not difficult to arrive at a relation for the currents in then presence of the particle and without it:
(5)
Let us illustrate the technique on the example of a capillary with the radius of the hole 100 nm (Fig.1). When calculating for a particle with rчастицы ≅ 50 nm, we obtain Rпри/Rбез ≈ 2, the resistance value doubles, which entails a 50% drop in the current value!
In the nanocapillary printing process, when the capillary is close to the sample surface and the hopping mode of its movement is switched off, this drop will be easily detected by the feedback system, allowing accurate counting during localised nanoparticle deposition.
This registration is implemented in the of the FemtoScan X Ion scanning capillary microscope control software [18], which registers corresponding signal changes from the amplifier flowing through the aperture of the ion current capillary.
The scanning capillary microscope can also be used for local application of tiny liquid droplets on the substrate or in the sample area [19]. The mechanism of operation is illustrated in Fig.2. Due to the capillary effect (Laplace’s law), a microdrop is formed at the tip of the capillary filled with electrolyte, which, when the capillary is brought to a conducting substrate, contacts it and a current starts to flow between the electrode in the capillary medium and the substrate electrode. The appearance of current is detected by feedback system, which allows to stop convergence and withdraw the capillary, while the droplet remains on the surface due to wetting.
This technique was also implemented on the FemtoScan X Ion scanning capillary microscope platform [20]. The use of SCM in this case is due to the above-described mechanism and the need for local and metered application of micrometre-period droplets on the horizontal surface of the sample (substrate). The piezo manipulator, which provides precision movement of the substrate, is an excellent help.
Saline solution (0.9 M NaCl) was taken as applied liquid, the gold plated MicroSD/SD adapter contact served as the conductive substrate. The feedback system ability to register current at contact of a drop with the substrate and stop the supply without breaking the capillary was noted.
A capillary with an orifice size of about 1.5 µm was used in this work, and the linear dimensions of the drop formed at its tip are of a similar order of magnitude (Fig.3), which makes it possible to apply drops with a volume of the order of femtolitres (µm3 = 10-18 m3 = 10-15 litres). The current value at the contact with the substrate was 6.5 nA.
There are various ways to control the applied drop volume. The first is to select a capillary with a specific outlet size. Another method involves the use of external pressure applied by a compressor system through the upper free opening of the capillary. Electrophoresis, directed transfer of charged particles under the electric field influence can also be used to vary delivery of the substance.
CONCLUSIONS
Intracellular delivery is a key step in modern biological research and paves the way for numerous biomedical discoveries. Nowadays, delivery of substances into cells is becoming increasingly important in industrial and medical applications ranging from biomanufacturing to cell therapy.
Therefore, scanning capillary microscopy acquires special interest as a method of morphology research, measurement of local biocurrents and as a method of targeted delivery of substances, which allows us to study reaction of cells to external influence and, as a consequence, to obtain qualitatively new and more significant information about their vital activity process.
The uniqueness of capillary-assisted delivery of molecules, proteins and DNA lies in its accessibility, applicability to different cell types with minimal damage to the cell membrane and relative rapidity. This method can be used for efficient delivery of advanced therapeutics (nanoinjection) and for non-destructive sampling (nanobiopsy), with immediate information on changes in cell phenotype due to manipulation.
However, successful implementation of this technology requires knowledge of several scientific fields, as well as wide dissemination of scanning capillary microscopy to anyone interested in intracellular delivery: biologists studying cell mechanics and morphology, chemists developing new substances, cellular physiologists and biotechnologists studying the delivery mechanisms of substances into the cell.
ACKNOWLEDGEMENTS
This work was performed under the state order with the financial support of the Physical Department of Lomonosov Moscow State University (Registration subject 122091200048-7). FemtoScan Online software is provided by "Advanced Technologies Center", www.nanoscopy.ru. Special thanks to T.O. Sovetnikov for support the Basis Foundation (Contract No, 24-2-10-9-1).
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
The cell is endowed with a dynamic mechanism, and access to it is strictly regulated by intracellular processes. Cells transmit information to each other through molecules, DNA encodes RNA and proteins, proteins transmit signals and act as building blocks of cellular structure, lipids form the membrane and store energy. Traditional methods of substance delivery can interfere with cellular function, limiting their applicability. Introducing molecules and substances into cells is an important experimental step in understanding its function, in controlling the cell and reprogramming its behaviour.
There are different ways to deliver substances into mammalian cells: by lipid vesicles and virus-like particles, by chemical transfection, electroporation and micro- and nano-injections.
Nanoinjection is an intracellular delivery process using nano-needles [1-3]. Nano-needles are generally not harmful to cells, including neurons, cardiomyocytes and immune cells [4-6]. Nanoinjection increases efficiency of plasmid DNA (pDNA) transfection in hard-to-transfect cells [7].
Not only nano-needles can be used for injection, but also glass capillaries with micron-sized exit hole diameters can be used to mechanically penetrate the plasma membrane, which also allows delivery of molecules. However, the large size of the capillary tip (typically 0.5 to 75 µm in outer diameter) relative to the cell size can deform the membrane during injection, disrupting the actin cytoskeleton and altering the cell morphology.
Using a puller (e.g., P2000, Sutter Instruments), capillaries with a minimum exit hole radius of up to 10 nm can be fabricated. Using an electrode placed in the capillary, the transport of molecules can be monitored by applying a suitable voltage and measuring the ionic current. The polarity and voltage value depend on the charge of the delivered molecule, and the voltage value should not exceed ±1 V [8, 9]. Moreover, the small size of the capillary tip relative to the cell allows not to deform the cell membrane. Translocation of one macromolecule through the capillary tip results in a detectable change in the measured ionic current, which can be used to characterise and quantify the number of molecules passing through the capillary [10, 11].
In [12], a platform is described that uses capillaries to deliver specific amounts of macromolecules into cultured cell lines and primary cells with resolution at the single molecule level. The capillary is used both as a probe of a scanning capillary microscope and as a needle for injection. The SCM is used to settle over the cell surface before the capillary is inserted to a predetermined depth into a specific location in the cell. With this setup, the quantitative delivery of DNA, globular proteins and protein fibrils into cells has been demonstrated with resolution at the single molecule level. And it is also demonstrated that delivery leads to a change in the phenotype of the cell.
The results on application of scanning capillary microscopy for local delivery of nanoparticles are also known [13, 14]. In this regard, the question of calculating their number that has passed through the holes of the nanocapillary arises. This approach will allow not only local but also high-precision delivery of nanoparticles to the cell surface.
EXPERIMENTAL RESEARCH
In order to develop the nanocapillary printing techniques, our group present a technique for counting the deposited particles number. The idea consists in selecting a capillary opening commensurate with the particles to be delivered (deposited), in which case the nanoparticle exit from the capillary (under the action of the applied potential difference) will be accompanied by the forming of a kind of barrier to the passage of ions, the value of the ionic current will decrease.
The known results [15-17] on the current behaviour description through the nanocapillary aperture show that over a wide range of end aperture size, the current through it can be described by Ohm’s law. The magnitude of the ionic current flowing through the aperture in the capillary is determined by the expression:
(1)
where – saturation current (away from the sample surface), z – sample surface distance, А – coefficient, taking into account the capillary geometry, determined through the relation [17]:
(2)
where – aperture outer diameter, – aperture inner diameter, L – distance from the capillary end to the electrode inside the capillary. The saturation current flowing through the nanocapillary at a large distance from the sample is mainly determined through the capillary resistance Rp via Ohm’s law:
(3)
where U – electrode voltage.
For resistance of the nanocapillary the following relation is valid:
(4)
where – conductivity of electrolyte medium, – inner radius of a capillary, – capillary apex angle (on average from 3° to 5°).
Using also the expressions for determining the size and resistance of the capillary (3, 4) and the current near the surface (1), and neglecting the constant components, it is not difficult to arrive at a relation for the currents in then presence of the particle and without it:
(5)
Let us illustrate the technique on the example of a capillary with the radius of the hole 100 nm (Fig.1). When calculating for a particle with rчастицы ≅ 50 nm, we obtain Rпри/Rбез ≈ 2, the resistance value doubles, which entails a 50% drop in the current value!
In the nanocapillary printing process, when the capillary is close to the sample surface and the hopping mode of its movement is switched off, this drop will be easily detected by the feedback system, allowing accurate counting during localised nanoparticle deposition.
This registration is implemented in the of the FemtoScan X Ion scanning capillary microscope control software [18], which registers corresponding signal changes from the amplifier flowing through the aperture of the ion current capillary.
The scanning capillary microscope can also be used for local application of tiny liquid droplets on the substrate or in the sample area [19]. The mechanism of operation is illustrated in Fig.2. Due to the capillary effect (Laplace’s law), a microdrop is formed at the tip of the capillary filled with electrolyte, which, when the capillary is brought to a conducting substrate, contacts it and a current starts to flow between the electrode in the capillary medium and the substrate electrode. The appearance of current is detected by feedback system, which allows to stop convergence and withdraw the capillary, while the droplet remains on the surface due to wetting.
This technique was also implemented on the FemtoScan X Ion scanning capillary microscope platform [20]. The use of SCM in this case is due to the above-described mechanism and the need for local and metered application of micrometre-period droplets on the horizontal surface of the sample (substrate). The piezo manipulator, which provides precision movement of the substrate, is an excellent help.
Saline solution (0.9 M NaCl) was taken as applied liquid, the gold plated MicroSD/SD adapter contact served as the conductive substrate. The feedback system ability to register current at contact of a drop with the substrate and stop the supply without breaking the capillary was noted.
A capillary with an orifice size of about 1.5 µm was used in this work, and the linear dimensions of the drop formed at its tip are of a similar order of magnitude (Fig.3), which makes it possible to apply drops with a volume of the order of femtolitres (µm3 = 10-18 m3 = 10-15 litres). The current value at the contact with the substrate was 6.5 nA.
There are various ways to control the applied drop volume. The first is to select a capillary with a specific outlet size. Another method involves the use of external pressure applied by a compressor system through the upper free opening of the capillary. Electrophoresis, directed transfer of charged particles under the electric field influence can also be used to vary delivery of the substance.
CONCLUSIONS
Intracellular delivery is a key step in modern biological research and paves the way for numerous biomedical discoveries. Nowadays, delivery of substances into cells is becoming increasingly important in industrial and medical applications ranging from biomanufacturing to cell therapy.
Therefore, scanning capillary microscopy acquires special interest as a method of morphology research, measurement of local biocurrents and as a method of targeted delivery of substances, which allows us to study reaction of cells to external influence and, as a consequence, to obtain qualitatively new and more significant information about their vital activity process.
The uniqueness of capillary-assisted delivery of molecules, proteins and DNA lies in its accessibility, applicability to different cell types with minimal damage to the cell membrane and relative rapidity. This method can be used for efficient delivery of advanced therapeutics (nanoinjection) and for non-destructive sampling (nanobiopsy), with immediate information on changes in cell phenotype due to manipulation.
However, successful implementation of this technology requires knowledge of several scientific fields, as well as wide dissemination of scanning capillary microscopy to anyone interested in intracellular delivery: biologists studying cell mechanics and morphology, chemists developing new substances, cellular physiologists and biotechnologists studying the delivery mechanisms of substances into the cell.
ACKNOWLEDGEMENTS
This work was performed under the state order with the financial support of the Physical Department of Lomonosov Moscow State University (Registration subject 122091200048-7). FemtoScan Online software is provided by "Advanced Technologies Center", www.nanoscopy.ru. Special thanks to T.O. Sovetnikov for support the Basis Foundation (Contract No, 24-2-10-9-1).
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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