Method for loading biological particles with a molecule of interest

Abstract

A method for loading biological particles with a molecule of interest comprising selecting a microfluidic and nanofluidic chip device (1), mixing the biological particles with a solution comprising the molecule of interest, applying the mixed biological particles solution to a fluid inlet (2.1) of the chip device (1), applying a pressure to the chip device (1) forcing the mixed biological particles solution to pass through a inlet microchannel (4.1), then to a nanochannel (4.3) so that pressure on the biological particle cause the molecule of interest to enter the said biological particle and, then through an outlet microchannel (4.2), recovering the said loaded biological particles.

Description

[0001] METHOD FOR LOADING BIOLOGICAL PARTICLES WITH A MOLECULE OF INTEREST

[0002] Technical Field

[0003] The present invention relates to a microfluidic and nanofluidic chip device.

[0004] The present invention also relates to a method of fabricating the microfluidic and nanofluidic chip device according to the present invention.

[0005] The present invention also pertains to a use of the microfluidic and nanofluidic chip device according to the present invention to perform mechanoporation of biological particles surrounded by a lipid bilayer in pharmaceutical, GMP conditions.

[0006] The present invention also relates to a method for loading biological particles with a molecule of interest and to a method for loading biological particles with a molecule of interest by implementing the microfluidic and nanofluidic chip device according to the present invention.

[0007] State of the art

[0008] A microfluidic and nanofluidic chip device is a small-scale system designed to manipulate and control fluids at the micro (micrometer) and nano (nanometer) scales. These devices integrate tiny channels and structures that allow the precise handling of small volumes of liquids, gases, or even individual molecules.

[0009] A variety of nano-delivery systems is known, aiming to carry small molecules, nucleic acids, proteins, or other exogenous cargos into the body or focal lesion, which is not only helpful for the diagnosis and treatment of multiple diseases and pathology such as cancers, infectious diseases, inflammatory diseases, cardiovascular diseases, neurodegenerative diseases, etc., but are also used to improving the therapeutic impact of such systems.

[0010] In order to deliver exogenous cargos into the interior of biological particles, in particular lipid bilayer-delimited biological particles such as extracellular vesicles or even cells, commonly used methods include incubation and electroporation. The incubation method includes mixing the cargos with the biological particles uniformly under a certain temperature, and so the cargo molecules gradually diffuse into the interior of the biological particles along the concentration gradient thereby achieving cargo loading. Although the incubation method is simple and easy to operate, this method generally has a problem of low cargo-loading efficiency. Electroporation, as another commonly used cargo-loading method, includes stimulating the biological particles by applying an electric field so that some temporary pores would appear on the membrane surface. As such, the cargo molecules are transported into the biological particles through the pores. Then after a certain period of time, the lipid bilayer-delimited biological particles returns to their original state. In this way, the cargos are packaged in the biological particles, thus accomplishing the cargo-loading process. While this method improves the cargo-loading efficiency, the external electric field may however induce excessive damages on the lipid bilayer-delimited biological particles such as extracellular vesicles (EVs) and also on their carrying exogenous cargos.

[0011] An appropriate pharmaceutical, GMP conditions, process for the cargo loading of biological particles through mechanoporation should be as fast as possible and as effective as possible in terms of costs while being reliable and reproducible without a reduction of the quality of the process from batch-to-batch production.

[0012] Microfluidic chips have been used in cellular loading. In particular, EP401 1494 describes a microfluidic and nanofluidic chips that can be applied to the study of cargo loading of biological particles such as extracellular vesicles. Such loading is performed through a mechanoporation process. EP401 1494 describes a microfluidic and nanofluidic chip comprising a fluid input and a fluid output, at least one nanochannel layout comprising at least one nanochannel and at least one microchannel layout being alternately stacked. While this chips device can be used for the loading of exogenous cargos into extracellular vesicles, however in practise, this device is not convenient for pharmaceutical and good manufacturing practices (GMP) applications which is needed in order to use lipid bilayer-delimited biological particles loaded with exogeneous cargos in daily medical practises. Indeed, the inventors have found that the process of cargo loading of biological particles by implementing the chips device described in EP401 1494 encounters significant levels of dead volumes resulting in the waste of cell culture media, the waste of biological particles and exogeneous cargos, which can be very costly but also dead volumes can generate the presence of several clogging issues rendering it unreliable from batch-to-batch production. The clogging issues may also results in local concentration change of the biological particles and / or matters present in the solution leading to local microenvironment that can alter the efficiency and the uniformity of the loading of the biological particles giving rise to heterogeneous products and rendering the chips not suitable for GMP production of loaded biological particles. In addition, the presence of significant levels of dead volume is also not compatible with GMP manufacture since dead volume can generate long-term accumulation of matters, solutions, etc... that can lead to a deterioration and / or denaturation of the chips and which could also contaminate the loading environment of the biological particles. In addition, this chips device is also not really upscalable. Furthermore, this chip device is quite fragile with a significant level of breakage, resulting also in the waste of consumables while rendering it not really reliable from batch-to-batch production. The fragility of the chips is also increased as a consequence of the clogging issues generating an increase in the internal pressure within the chips and resulting in part from the presence of the significant presence of dead volumes. The significance level of breakage combined with the significant level of waste due in part to the presence of dead volumes when implementing the chips device in the process of cargo loading of biological particles renders this chips device not scalable and too expensive for pharmaceutical and good manufacturing practices (GMP) applications.

[0013] Amongst other documents, US7070684 describes a microsystem for manipulating particles suspended in a liquid using microelectrodes positioned on the walls of a channel, which generate an electric field barrier partially crossing the channel. The document US2014170679 relates in general to the field of particle detection and analysis and more particularly to particle detection and analysis using a fluidic chip. The document US2018292305 relates to devices for profiling particles, such as rare cells, in a flow. In particular, it relates to devices that use magnetism for profiling particles in a flow chamber.

[0014] While these documents praise the merits of their technology, in practice, there is a need to provide a microfluidic and nanofluidic chips device able to be implemented in a method for loading biological particles with a molecule of interest which is fast, cost-effective in order to be easily scalable and easily industrialised while being reliable from batch-to-batch production.

[0015] In particular, there is a need to provide a microfluidic and nanofluidic chips device generating few or even no dead volumes leading to a better cost- effective method for loading biological particles with a molecule of interest which is able to be applied for pharmaceutical and good manufacturing practices (GMP) applications. In addition, there is also a need to provide a microfluidic and nanofluidic chips device which is robust with a very few level of breakage rendering it scalable and compatible with high-intensive production of the pharmaceutical and GMP applications. Objectives of the invention

[0016] The present invention aims to overcome the drawbacks of the state of the art, in particular those described above.

[0017] In particular, the present invention proposes to provide a microfluidic and nanofluidic chips device generating, when implemented in a method for loading biological particles with a molecule of interest, few or even no dead volumes (in terms of media or solution, exogenous cargos and / or biological particles) leading to a more robust chips device and which more reliable leading to a more efficient, more reliable, and better cost-effective method able to be applied for pharmaceutical and good manufacturing practices (GMP) applications.

[0018] Summary of the invention

[0019] To achieve the aforementioned objectives, the present invention provides a microfluidic and nanofluidic chip device comprising: at least one fluid inlet comprising at least one fluid inlet hole, at least one fluid outlet comprising: o at least one fluid outlet hole, and o at least one fluid outlet chamber being delimited by a bottom wall, an upper wall, and at least a side wall connecting said bottom wall to said upper wall in a sealed manner, said fluid outlet hole being located within said bottom wall and / or said upper wall, at least one fluid module comprising: o a microchannel layout comprising at least one inlet microchannel having a constant section with its smallest dimension in the micrometer range, said inlet microchannel being connected to and in fluid communication with said fluid inlet, and at least one outlet microchannel having a constant section with its smallest dimension in the micrometer range, said outlet microchannel being connected to and in fluid communication with said fluid outlet chamber, o a nanochannel layout comprising a plurality of nanochannels, wherein each nanochannel is directly connected to and in fluid communication with said inlet microchannel and with said outlet microchannel, said plurality of nanochannels providing the sole fluid connection between said inlet microchannel and said outlet microchannel, each nanochannel having a constant section with its smallest dimension in the nanometer range, wherein said at least one inlet microchannel and said at least one outlet microchannel are alternatively stacked in a manner that said inlet microchannel is adjacent to said outlet microchannel, and wherein each nanochannel of the plurality of nanochannels having a first longitudinal end and a second longitudinal end, said ends being opposite to each other along a longitudinal axis of each nanochannel, and wherein said ends are directly connected to and in fluid communication either with said inlet microchannel and / or with said outlet microchannel, and wherein said fluid outlet chamber comprising at least one support column connecting said bottom wall to said upper wall, said support column being arranged to reinforce the structure of said fluid outlet chamber by maintaining a predetermined distance between said bottom wall and said upper wall.

[0020] Indeed, a microfluidic and nanofluidic chips device according to the present invention combines a fluid inlet, a fluid outlet, and a fluid module comprising at least a inlet microchannel, an outlet microchannel and a plurality of nanochannels as described. This device is centred on the fact that the longitudinal ends of each nanochannels are directly connected to and in fluid communication either with an inlet microchannel and / or with an outlet microchannel, and wherein the fluid outlet comprises a fluid outlet chamber having at least one support column between the bottom wall and the upper wall of the fluid outlet chamber. These combined technical characteristics allow a device with very few or even with no dead-ends of the nanochannel since the longitudinal ends of the nanochannels are connecting directly, in a fluid manner, either with the inlet microchannel and / or with the outlet microchannel. This results in a strong reduction of dead volumes when implementing the chips device in a process of cargo loading of biological particles through mechanoporation. This leads to a strong reduction of the waste in culture media, solution, exogenous cargos as well as biological particles, while also reducing the occurrence of clogging issues. In addition, the support column in the outlet chamber reinforces the structure of the device, giving a more robust device with a drastic reduction of breakage leading, even when pressure is applied, to a more reliable device giving a more constant quality from batch-to-batch production. The unique combination of the above characteristics results into a more robust and reliable microfluidic and nanofluidic chips device allowing a more efficient, fast (since less breakage is observed) and cost-effective production process of loaded biological particles with exogeneous cargos which is compatible with pharmaceutical, GMP, production. Since the chips device is more robust, reliable and cost-effective, the chips device according to the present invention is also compatible with a scalable production which may involve different pressure conditions, different tension on the structure of the device and / or different flow rate when implementing the device in a process of cargo loading of biological particles through mechanoporation.

[0021] Other embodiments of the microfluidic and nanofluidic chip device according to the present invention are indicated in the appended claims.

[0022] The present invention also relates to a method of fabricating the microfluidic and nanofluidic chip device according to the present invention, comprising:

[0023] - fabricating a plurality of nanochannels in a first substrate to obtain a nanochannel layout wherein each nanochannel having a constant section with its smallest dimension in the nanometer range;

[0024] - fabricating a fluid inlet and a fluid output in a second substrate,

[0025] - fabricating at least one inlet microchannel and at least one outlet microchannel in said second substrate wherein said fluid inlet is directly connected and in fluid communication with said at least one inlet microchannel and wherein said fluid outlet is directly connected and in fluid communication with said at least one outlet microchannel, wherein said at least one inlet microchannel and said at least one outlet microchannel are alternatively arranged at intervals to form a microchannel layout,

[0026] - alternately stacking and bonding at least one nanochannel layout and at least one microchannel layout to obtain the micro- and nano-fluidic chip, wherein the at least one inlet microchannel and the at least one outlet microchannel are fluidly connected through said plurality of nanochannels.

[0027] Other embodiments of the method of fabricating the microfluidic and nanofluidic chip device according to the present invention are indicated in the appended claims.

[0028] The present invention also relates to the use of the microfluidic and nanofluidic chip device described here above, to perform mechanoporation of biological particles surrounded by a lipid bilayer in pharmaceutical, GMP conditions.

[0029] Other embodiments of the use of the microfluidic and nanofluidic chip device according to the present invention are indicated in the appended claims.

[0030] The present invention also relates to a method for loading biological particles with a molecule of interest. Prior art methods for loading biological particles with a molecule of interest include various approaches using passive or active techniques such as microinjection, electroporation, or surface adsorption. However, these techniques have several limitations, including variable yields, risks of damage to the biological particles or their integrity, scalability constraints, and sometimes insufficient efficiency in controlling molecular loading. For instance, microinjection methods require precise control over injection and are often limited to relatively small volumes, making them difficult to apply on a large scale or to complex biological populations. Additionally, electroporation, while effective for certain cells or extracellular vesicles, may result in undesirable side effects such as membrane damage, EV aggregation as well as cargo aggregation, impacting EV integrity but also making the cargo devoid of any functionality.

[0031] These challenges highlight the need for improved loading processes, particularly in contexts where high loading levels are required while maintaining the biological integrity of the particles. Current solutions do not always offer reliable and reproducible results, necessitating the development of new methods and devices to optimize molecular loading in a microfluidic setting, allowing for precise control and enhanced efficiency.

[0032] In that context, the document Hao Rui et al. “A high-throughput nanofluidic device for exosome nanoporation to develop cargo delivery vehicles” Small, Vol 17, no. 35, 21 July 2021 (DOI: 10.1002 / smll.202102150) presents a high- throughput nanofluidic device designed to load cargo, specifically exosomes, with molecules of interest by utilizing microfluidic and nanofluidic channels. However, while praising the merits of its technology, this document does not offer a reliable method with consistent and efficient loading across batches, especially when scaled up for larger industrial applications.

[0033] As a consequence, there is still a need to provide a method that ensures consistent, reproducible, and scalable loading of biological particles with molecules of interest, particularly when considering industrial-scale production, to enhance efficiency and maintain biological integrity across diverse particle types.

[0034] The method for loading biological particles with a molecule of interest according to the present invention comprises the steps of: selecting a microfluidic and nanofluidic chip device, preferably the microfluidic and nanofluidic chip device according to the present invention, comprising an fluid inlet and an fluid outlet, one or several (identical) inlet microchannels and outlet microchannels having a constant section with its (their) smallest dimension in the micrometer range, and a plurality of identical nanochannels having a constant section with their smallest dimension in the nanometer range wherein the nanochannels provide the sole fluid connection between said inlet microchannel and said outlet microchannel, mixing the said biological particles with a solution comprising the said molecule of interest, applying the mixed biological particles solution with the said molecule of interest to the fluid inlet of the said microfluidic and nanofluidic chip device, applying a pressure to the said microfluidic and nanofluidic chip device forcing the biological particles and the molecule of interest to pass through a inlet microchannel, then to a nanochannel so that pressure on the biological particle and / or physical forces cause the molecule of interest to enter the said biological particle and, then through an outlet microchannel, wherein, preferably, at least 95% v / v, more preferably at least 98% v / v or even more preferably 99% v / v, of the mixed biological particles solution with the said molecule of interest entering through said fluid inlet is passing through said fluid outlet, recovering the said loaded biological particles.

[0035] Indeed, the method for loading biological particles with a molecule of interest according to the present invention is centered on the fact that nanochannels provide the sole fluid connection between said inlet microchannel and said outlet microchannel and on the fact that at least 95% v / v, more preferably at least 98% v / v or even more preferably 99% v / v, of the mixed biological particles solution with the said molecule of interest entering through said fluid inlet is passing through said fluid outlet (reflecting the almost total absence of dead volumes). This permits the loading of biological particles with the molecule of interest at a high efficiency rate. In addition, the fact that at least 95% v / v, more preferably at least 98% v / v or even more preferably 99% v / v, of the mixed biological particles solution with the said molecule of interest entering through said fluid inlet is passing through said fluid outlet provides a method for loading biological particles with a molecule of interest by creating only very limited, or even no, dead volumes within the microfluidic and nanofluidic chip device. The unique combination of the characteristics of the method according to the present invention allows having a method for loading biological particles with a molecule of interest that is compatible with GMP production requirements since the method allows producing loaded biological particles with a molecule of interest of high-quality, efficient, and uniform from batch-to-batch production by avoiding clogging issues and by avoiding micro-environment perturbations that may arise in the presence of significant levels of dead volumes.

[0036] Other embodiments of the method for loading biological particles with a molecule of interest according to the present invention are indicated in the appended claims.

[0037] The present invention also relates to a method for loading biological particles with a molecule of interest by implementing the microfluidic and nanofluidic chip device according to the present invention, said method comprising the steps of: selecting said microfluidic and nanofluidic chip device according to the present invention, mixing the said biological particles with a solution comprising the said molecule of interest, applying the mixed biological particles solution with the said molecule of interest to the fluid inlet of the said microfluidic and nanofluidic chip device, applying a pressure to the said microfluidic and nanofluidic chip device forcing the biological particles and the molecule of interest to pass through said microchannel layout comprising at least one inlet microchannel, then to said nanochannel layout comprising a plurality of nanochannels so that pressure on the biological particle and / or physical forces cause the molecule of interest to enter the said biological particle and then through said at least one outlet microchannel in order to reach said fluid outlet, recovering the said loaded biological particles.

[0038] Other embodiments of the method for loading biological particles with a molecule of interest by implementing the microfluidic and nanofluidic chip device according to the present invention are indicated in the appended claims.

[0039] The present invention also provides an assembly compatible with scalable Good Manufacturing Practice (GMP) production comprising a plurality of microfluidic and nanofluidic chip devices according to the present invention, wherein the chip devices are arranged in parallel to each other, and wherein the assembly further comprises: a common inlet distribution system configured to supply each chip device with fluid under homogeneous and controlled flow conditions, the common inlet distribution system being connected to each fluid inlet of each microfluidic and nanofluidic chip device, a common or individually addressable outlet collection system configured to retrieve processed fluids from each chip device, the common or individually addressable outlet collection system being connected to each fluid outlet of each microfluidic and nanofluidic chip device, optionally, a supporting frame or module configured to maintain the relative positioning of the chip devices, such that the parallel arrangement of the chip devices enables linear upscaling of processing throughput while preserving the individual fluidic performance of each microfluidic and nanofluidic chip device.

[0040] Indeed, the assembly of multiple microfluidic and nanofluidic chip devices arranged in parallel offers significant advantages in terms of scalability, robustness, and regulatory compliance. By operating several identical chips simultaneously, the assembly enables a linear and predictable increase in processing throughput without altering the fluidic behavior or performance of each individual device. This modular parallelization mitigates the limitations of single-chip operation, such as restricted flow capacity or limited production volume, and allows reliable upscaling while preserving the precision and uniformity inherent to micro- and nanofluidic architectures. Furthermore, integrating the chips within a common distribution and collection manifold simplifies fluid handling, reduces system variability, and ensures homogeneous operating conditions across all units. The structural frame maintaining sterile boundaries and controlled chip positioning facilitates implementation under GMP standards, supporting reproducible large-scale manufacturing and seamless transition from laboratory-scale operation to industrial production environments.

[0041] Other embodiments of the assembly according to the present invention are indicated in the appended claims.

[0042] Detailed description of the invention :

[0043] Other features and advantages of the present invention will be derived from the following non-limiting description, and with reference to the following figure and examples. Figure 1 shows a schematic longitudinal section of a first embodiment of the microfluidic and nanofluidic chip device of the according to the present invention.

[0044] Figure 2 shows an enlarged view of the longitudinal section of the fluid inlet and the fluid outlet of the first embodiment of the microfluidic and nanofluidic chip device of the according to the present invention.

[0045] Figure 3 shows a schematic representation of the microfluidic and nanofluidic chip device according to the prior art (EP401 1494) together with a pressure profile in an operational mode within this microfluidic and nanofluidic chip device according to the prior art.

[0046] Figure 4 shows a schematic representation of the microfluidic and nanofluidic chip device according to the present invention together with a pressure profile in an operational mode within this microfluidic and nanofluidic chip device according to the present invention.

[0047] Figure 5 illustrates the loading efficiency achieved with the method of the present invention (EX5) and with a prior art method (CE1 ) as compared to the quantity of the most abundant natural miRNAs found in EVs from Wharton’s Jelly cells.

[0048] Figure 6 illustrates a dose response curve of EVs loaded with miR140 using the method of the present invention compared with EVs produced using a prior art method.

[0049] According to the present invention, preferably, the term "extracellular vesicle (EV)” is understood to mean lipid bilayer-delimited particles that can be secreted by numerous cell types. The size of the extracellular vesicles ranges from 20 nm to more than 1000 nm, wherein exosomes represent extracellular vesicles with a size range between 30 and 150 nm, while microvesicles may have a size ranging from 100 nm to 1000 nm and apoptotic bodies have a size greater than 1000 nm, such as for example exosomes, ectosome, microvesicle and / or exosome-like vesicles. Alternatively or preferably, the term "extracellular vesicle (EV)” is understood to mean lipid bilayer- delimited particles which include at least one membrane vesicles secreted by a cell such as exosomes, subcellular particles with a size of 30 nm to 2000 nm and having a membrane structure, cell membrane nanoparticles with a size of 30 nm to 2000 nm, artificially synthesized nanoparticles with a size of 30 nm to 2000 nm wrapped in a phospholipid bilayer, liposomes with a diameter of 30 nm to 2000 nm, or viral vectors, etc. Alternatively, or in addition, according to the present invention, preferably, the term ‘extracellular vesicle”, EV, means particles that are released from cells, are delimited by a lipid bilayer, and cannot replicate on their own. Alternatively, or in addition, according to the present invention, preferably, the term “exosomes” means a subtype of small extracellular vesicles originating from the endosomal system.

[0050] According to the present invention, preferably, the term “biological particles” is understood to mean extracellular vesicles, artificially synthesized nanoparticles, cell membrane nanoparticles, eukaryotic cells and / or prokaryotic cells.

[0051] According to the present invention, preferably, the term “molecule of interest” is understood to mean a molecule able to have a therapeutic and / or a prophylactic impact on a particular pathology and / or disease. Preferably, the molecule of interest is a peptide, protein, shRNA, miRNA, mRNA, gRNA, pri-miRNA, pre-miRNA, circular RNA, piRNA, tRNA, rRNA, snRNA, IncRNA (long non-coding RNA), ribozymes, mini-circle DNA, plasmid DNA, circular RNA, siRNA, agomir, antagomir or a combination thereof.

[0052] According to the present invention, preferably, the term “mechanoporation” is understood to mean a passage of the biological particles that have been harvest through the nanochannel layout so that the membrane of the biological particles is mechanically squeezed and / or is compressed by the passage through said nanochannel layout preferably leading to the formation of holes in the membrane of the extracellular vesicles allowing the molecule of interest to enter into the extracellular vesicles.

[0053] Figure 1 shows a first embodiment of the microfluidic and nanofluidic chip device 1 according to the present invention comprising: at least one fluid inlet 2 comprising at least one fluid inlet hole 2.1 , at least one fluid outlet 3 comprising: o at least one fluid outlet hole 3.1 , and o at least one fluid outlet chamber 3.2 being delimited by a bottom wall, an upper wall, and at least a side wall connecting said bottom wall to said upper wall in a sealed manner, said fluid outlet hole being located within said bottom wall and / or said upper wall, at least one fluid module 4 comprising: o a microchannel layout comprising at least one inlet microchannel 4.1 having a constant section with its smallest dimension in the micrometer range, said inlet microchannel 4.1 being connected to and in fluid communication with said fluid inlet 2, and at least one outlet microchannel 4.2 having a constant section with its smallest dimension in the micrometer range, said outlet microchannel 4.2 being connected to and in fluid communication with said fluid outlet chamber 3.2, o a nanochannel layout comprising a plurality of nanochannels 4.3, wherein each nanochannel 4.3 is directly connected to and in fluid communication with said inlet microchannel 4.1 and with said outlet microchannel 4.2, said plurality of nanochannels 4.3 providing the sole fluid connection between said inlet microchannel 4.1 and said outlet microchannel 4.2, each nanochannel 4.3 having a constant section with its smallest dimension in the nanometer range, wherein said at least one inlet microchannel 4.1 and said at least one outlet microchannel 4.2 are alternatively stacked in a manner that said inlet microchannel 4.1 is adjacent to said outlet microchannel 4.2, and wherein each nanochannel 4.3 of the plurality of nanochannels 4.3 having a first longitudinal end 4.31 and a second longitudinal end 4.32, said ends 4.31 , 4.32 being opposite to each other along a longitudinal axis L2 of each nanochannel 4.3, and wherein said ends 4.31 , 4.32 are directly connected to and in fluid communication either with said inlet microchannel

[0054] 4.1 and / or with said outlet microchannel 4.2, and wherein said fluid outlet chamber 3.2 comprising at least one support column 3.3 connecting said bottom wall to said upper wall, said support column 3.3 being arranged to reinforce the structure of said fluid outlet chamber 3.2 by maintaining a predetermined distance between said bottom wall and said upper wall.

[0055] Preferably and according to the first embodiment of the microfluidic and nanofluidic chip device, the fluid inlet 2 further comprises at least one fluid inlet chamber

[0056] 2.2 being delimited by a bottom wall, an upper wall, and at least a side wall connecting said bottom wall to said upper wall in a sealed manner, said fluid inlet hole 2.1 being located within said bottom wall and / or said upper wall, wherein said fluid inlet chamber

[0057] 2.2 comprising at least one support column 2.3 connecting said bottom wall to said upper wall, said support column 2.3 being arranged to reinforce the structure of said fluid inlet chamber 2.2 by maintaining a predetermined distance between said bottom wall and said upper wall. Preferably, said fluid inlet chamber 2.2 and said outlet chamber 3.2 comprise a plurality of support columns 2.3, 3.3 connecting the bottom wall to the upper wall of the inlet chamber and outlet chamber respectively, the plurality of support columns 2.3, 3.3 being arranged to reinforce the structure of said fluid inlet and outlet chamber 2.2, 3.2 by maintaining a predetermined distance between said bottom wall and said upper wall. Indeed, the support column 2.3, 3.3 within the inlet chamber 2.2 (and also the outlet chamber 3.2) is arranged to reinforce the structural strength of the chamber in order for example to avoid collapse of the inlet 2.2 and / or outlet chamber 3.2 during the fabrication of the microfluidic and nanofluidic chip device 1 according to the present invention.

[0058] Figure 2 shows an enlarged view of the longitudinal section of the fluid inlet 2 (FIG. 2A) and the fluid outlet 3 (FIG. 2B) of the first embodiment of the microfluidic and nanofluidic chip device of the according to the present invention, wherein preferably, the fluid outlet chamber 3.2 has a first outlet chamber part 3.4 and a second outlet chamber part 3.5 and has a flared shape widening along a longitudinal axis LI of said fluid outlet chamber 3.2 from a first end to a second end of said fluid outlet chamber 3.2, wherein said outlet hole 3.1 is located within said first outlet chamber part 3.4 of said fluid outlet chamber 3.2, said first outlet chamber part 3.4 being located nearer said first end of said fluid outlet chamber 3.2 than said second outlet chamber part 3.5, while said outlet microchannel 4.2 being connected to and in fluid communication with said second outlet chamber part 3.5, said second outlet chamber part 3.5 being located nearer said second end of said fluid outlet chamber 3.2 than said first outlet chamber part 3.4, and / or the fluid inlet chamber 2.2 having a first inlet chamber part 2.4 and a second inlet chamber part 2.5 and having a flared shape widening along a longitudinal axis LI of said fluid inlet chamber 2.2 from a first end to a second end of said fluid inlet chamber 2.2, wherein said inlet hole 2.1 is located within said first inlet chamber part 2.4 of said fluid inlet chamber 2.2, said first inlet chamber part 2.4 being located nearer said first end of said fluid inlet chamber 2.2 than said second inlet chamber part 2.5, while said inlet microchannel 4.1 being connected to and in fluid communication with said second inlet chamber part 2.5, said second inlet chamber part 2.5 being located nearer said second end of said fluid inlet chamber 2.2 than said first inlet chamber part 2.4. For the sake of clarity, the vertical dashed line 2.6 in FIG. 2A is a longitudinal cross section of an “artificial” plane separating the first inlet chamber part 2.4 and the second inlet chamber part 2.5. Similarly, for the sake of clarity, the vertical dashed line 3.6 in FIG. 2B is a longitudinal cross section of an “artificial” plane separating the first outlet chamber part 3.4 and the second outlet chamber part 3.5. Preferably, the outlet hole 3.1 is located in the first outlet chamber part 3.4 at a first end, preferably at a vertex of a triangle in a longitudinal cross section of the fluid outlet 3. Preferably, the inlet hole 2.1 is located in the first inlet chamber part 2.4 at a first end, preferably at a vertex of a triangle in a longitudinal cross section of the fluid inlet 2. According to the first embodiment of the microfluidic and nanofluidic chip device, the fluid outlet chamber 3.2 has a first outlet chamber part 3.4 and a second outlet chamber part 3.5 and having a flared shape widening along a longitudinal axis LI of said fluid outlet chamber 3.2 from a first end to a second end of said fluid outlet chamber 3.2, wherein said outlet hole 3.1 is located within said first outlet chamber part 3.4 of said fluid outlet chamber 3.2, said first outlet chamber part 3.4 being located nearer said first end of said fluid outlet chamber 3.2 than said second outlet chamber part 3.5, while said outlet microchannel 4.2 being connected to and in fluid communication with said second outlet chamber part 3.5, said second outlet chamber part 3.5 being located nearer said second end of said fluid outlet chamber 3.2 than said first outlet chamber part 3.4, and the fluid inlet chamber 2.2 having a first inlet chamber part 2.4 and a second inlet chamber part 2.5 and having a flared shape widening along a longitudinal axis LI of said fluid inlet chamber 2.2 from a first end to a second end of said fluid inlet chamber 2.2, wherein said inlet hole 2.1 is located within said first inlet chamber part 2.4 of said fluid inlet chamber 2.2, said first inlet chamber part 2.4 being located nearer said first end of said fluid inlet chamber 2.2 than said second inlet chamber part 2.5, while said inlet microchannel 4.1 being connected to and in fluid communication with said second inlet chamber part 2.5, said second inlet chamber part 2.5 being located nearer said second end of said fluid inlet chamber 2.2 than said first inlet chamber part 2.4.

[0059] Preferably, the fluid module 4 comprises said nanochannel layout comprising a plurality of nanochannels 4.3 having each a longitudinal axis L2 substantially parallel to each other, said nanochannel layout being located between an inlet microchannel layout comprising at least one inlet microchannel 4.1 , preferably a plurality of inlet microchannels 4.1 , and an outlet microchannel layout comprising a plurality of outlet microchannels 4.2, said at least one inlet microchannel 4.1 , or preferably said plurality of inlet microchannels 4.1 , and said plurality of outlet microchannels 4.2 having each a longitudinal axis LI substantially parallel to each other and oriented substantially perpendicular to said longitudinal axis L2 of said plurality of nanochannels.

[0060] Preferably, each nanochannel 4.3 of the plurality of nanochannels (4.3) has a first longitudinal end 4.31 and a second longitudinal end 4.32, said ends (4.31 , 4.32) being opposite to each other along the longitudinal axis L2 of each nanochannel, and wherein said first longitudinal end 4.31 of each nanochannel 4.3 is directly connected to and in fluid communication with the first inlet microchannel 4.1 of the plurality of inlet microchannels 4.1 or the first outlet microchannel 4.2 of the plurality of outlet microchannels 4.2 while said second longitudinal end 4.32 of each nanochannel 4.3 is directly connected to and in fluid communication with the last inlet microchannel 4.1 of the plurality of inlet microchannel 4.1 or the last outlet microchannel 4.2 of the plurality of outlet microchannels 4.2. Preferably, each nanochannel 4.3 of the plurality of nanochannels (4.3) has a first longitudinal end 4.31 and a second longitudinal end 4.32, said ends (4.31 , 4.32) being opposite to each other along the longitudinal axis L2 of each nanochannel, and wherein said first longitudinal end 4.31 of each nanochannel 4.3 is directly connected to and in fluid communication with an inlet microchannel 4.1 of the plurality of inlet microchannels 4.1 or with an outlet microchannel 4.2 of the plurality of outlet microchannels 4.2 while said second longitudinal end 4.32 of each nanochannel 4.3 is directly connected to and in fluid communication with an inlet microchannel 4.1 of the plurality of inlet microchannel 4.1 or with an outlet microchannel 4.2 of the plurality of outlet microchannels 4.2.

[0061] Preferably, each nanochannels 4.3 of said plurality of nanochannels 4.3 has a longitudinal axis L2 substantially parallel to each other and wherein the central longitudinal axis of two adjacent nanochannels 4.3 of the plurality of nanochannels 4.3 are located at a distance comprised between 10 m and 500 pm from each other. Preferably, each nanochannels 4.3 of said plurality of nanochannels 4.3 are substantially parallel to each other and wherein two adjacent nanochannels 4.3 of the plurality of nanochannels 4.3 are located at a distance comprised between 10 pm and 500 pm from each other.

[0062] Preferably, the at least one inlet microchannel 4.1 has a constant section with its smallest dimension comprised between 1 pm and 1000 pm, more preferably between 40 pm and 500 pm, and / or wherein said at least one outlet microchannel 4.2 having a constant section with its smallest dimension comprised between 1 pm and 1000 pm, more preferably between 40 pm and 500 pm.

[0063] Preferably, each nanochannel 4.3 of the plurality of nanochannels 4.3 has a constant section with its smallest dimension comprised between 50 nm and 2000 nm. Alternatively, or preferably, each nanochannel 4.3 of the plurality of nanochannels 4.3 has a constant section with its smallest dimension comprised between 20% and 1000% of the smallest dimension of a section of an extracellular vesicle (smallest dimension of the constant section of the nanochannel / smallest dimension of a section of an extracellular vesicle passing through said nanochannel). Preferably the section of the channels are circular, substantially circular or elliptic (wherein the major axis is less than two-fold longer than the minor axis).

[0064] Preferably, the fluid module 4 has a length in said longitudinal axis LI comprised between 15 mm and 80 mm and a width in said longitudinal axis L2 comprised between 10 mm and 30 mm.

[0065] Preferably, the microfluidic and nanofluidic chip device according to the present invention is operatively connected to at least one pump, said pump being arranged for applying a pressure to the microfluidic and nanofluidic chip device 1 forcing the biological particles and the molecule of interest to pass through a inlet microchannel 4.1, then to a nanochannel 4.3 so that pressure on the biological particle and / or physical forces cause the molecule of interest to enter the said biological particle and, then through an outlet microchannel 4.2. Preferably, said at least one pump is a metering or a positive-displacement pump arranged for working at high pressure and preferably connected to several pistons so as to be able to work in continuous operation.

[0066] Preferably, the microfluidic and nanofluidic chip device according to the present invention and said at least one pump is operatively connected to a controller, preferably an automatic controller, arranged to control the passage of the microfluidic and nanofluidic chip device and of said at least one pump (i) from at least a STOP mode, wherein a pressure is not applied into said microfluidic and nanofluidic chip device 1, to at least one operational model, wherein a pressure is applied within the microfluidic and nanofluidic chip device 1 forcing the biological particles and the molecule of interest to pass through the device, and (ii) from said at least one operational mode to said at least one STOP mode.

[0067] Preferably, in the method for loading biological particles with a molecule of interest according to the present invention, more than 108biological particles per milliliter are mixed with the solution comprising the molecule of interest to form a mixed solution.

[0068] Preferably, the mixed solution comprises a phosphate-buffered saline (PBS), normal saline, Tris, Tris saline, or HEPES buffer.

[0069] Preferably, the flow velocity inside the microfluidic and nanofluidic chip device is equal or superior to 0.01 meter per second. More preferably, the flow velocity inside the microfluidic and nanofluidic chip device is comprised between 0.01 and 50 meter per second. Preferably, the flow rate within the microfluidic and nanofluidic chip device is equal or superior to 100 l / minute. More preferably, the flow rate within the microfluidic and nanofluidic chip device is comprised between 100 and 2000 pl / minute, even more preferably between 500 and 2000 l / minute.

[0070] Preferably, the mixed solution is washed of any potentially interfering impurities by passing (i) through a membrane filter, preferably with pores having a size equal to or smaller than 0,45 pm, more preferably equal to or smaller than 0,2 pm and / or (ii) by centrifugation at relative centrifugal forces between 2.000 g and 30.000 g, for a duration of at least 30 minutes and preferably around 120 minutes.

[0071] Preferably, when the molecule of interest if a nucleotide, the excess of uncharged molecule of interest after passage through the microfluidic and nanofluidic device is removed by a nuclease incubation step. More preferably, the nuclease incubation step having a duration between 30 minutes and 24 hours, preferably between 1 and 4 hours, more preferably 2 hours. More preferably, the nuclease incubation step is performed at a temperature comprised between 20°C and 40°C, preferably between 25°C and 35°C. More preferably, the nuclease incubation step is performed being performed at a nuclease concentration comprised between l U / mL and 50U / mL, preferably between l OU / mL and 20U / mL. More preferably , nuclease incubation step is performed with a Mg2+ concentration between 0,5 mM and 10 mM, preferably between 1 and 3 mM. More preferably, the excess nuclease after the nuclease incubation step is removed by diafiltration or dilution through a filter membrane with molecular weight cutoff between 100 kDA and 750 kDA, preferably 300 kDA.

[0072] Preferably, the molecule of interest is an oligonucleotide or a polynucleotide.

[0073] Preferably, the oligonucleotide is at a concentration between 1 nM and 1 mM.

[0074] Preferably, the oligonucleotide is at least a miRNA and / or a circular RNA.

[0075] Preferably, the method for loading biological particles with a molecule of interest according to the present invention further comprises a final step of treating the loaded biological particles with a solution comprising a nuclease so as to remove noninternalized nucleic acid molecules.

[0076] More preferably, the method for loading biological particles with a molecule of interest according to the present invention further comprises the step of polishing the composition comprising the loaded biological particles by filtration and / or centrifugation.

[0077] Preferably, the section of the nanochannels has one dimension comprised between 0.2 and 5 times the diameter of the biological particle.

[0078] Preferably, the biological particle is surrounded by a bilayer essentially consisting of phospholipids.

[0079] Preferably, the biological particle is an extracellular vesicle.

[0080] Preferably, the biological particle has a diameter or equivalent diameter comprised between 30 and 2000 nm, preferably between 50 and 1000 nm, more preferably between 100 and 500 nm.

[0081] Preferably, the pressure inside the microfluidic and nanofluidic chip device is set between 1 and 250 bar, preferably between 1 and 100 bars, more preferably between 5 and 50 bar, even more preferably between 10 and 30 bar.

[0082] Preferably, the assembly compatible with scalable Good Manufacturing Practice (GMP) production comprises at least 2, more preferably 3, 4, 5, 10, 20, 50, or even 100 microfluidic and nanofluidic chip devices according to the present invention, wherein the microfluidic and nanofluidic chip devices are arranged in parallel to each other. Indeed, parallel arrangement of these chip devices enables linear upscaling of processing throughput while preserving the individual fluidic performance of each microfluidic and nanofluidic chip device.

[0083] The present invention also provides a pharmaceutical composition comprising biological particles, preferably extracellular vesicles, preferably at least 1 x 1010(1010), loaded with at least one exogenous nucleic acid molecule, preferably an exogenous microRNA, and wherein the biological particles are loaded at an average level of at least 0.03, preferably at least 0.04 or even preferably at least 0.05 exogenous nucleic acid molecule per biological particle. Preferably, the biological particles loaded with the exogenous nucleic acid molecules are obtainable by implementing the method for loading biological particles with a molecule of interest according to the present invention or by using the device according to the present invention, or by implementing the method for loading biological particles with a molecule of interest by implementing the microfluidic and nanofluidic chip device according to the present invention.

[0084] Preferably, in this pharmaceutical composition, the biological particles are extracellular vesicles, preferably extracellular vesicles from immortalized mesenchymal stem cells such as Wharton’s Jelly cells, or from HEK cells. Preferably, in this pharmaceutical composition, the exogenous nucleic acid molecules are a miRNA and / or a circular RNA, preferably the miRNA is miR-140, miR-21 , and / or miR-146.

[0085] Examples. -

[0086] Example 1.- Microfluidic and nanofluidic chip device according to the prior art together with the pressure and flow rate that can be applied within said microfluidic and nanofluidic chip device according to the prior art in an operational mode.

[0087] Figure 3 shows a schematic representation of the microfluidic and nanofluidic chip device according to the prior art (EP401 1494) (see FIG. 3A). The microfluidic and nanofluidic chip device according to the prior art comprises one fluid inlet hole 2.1 , one fluid outlet hole 3.1 , and one fluid module 4 comprising: o a microchannel layout comprising a plurality of inlet microchannels, said plurality of inlet microchannel being connected to and in fluid communication with said fluid inlet hole 2.1 , and a plurality of outlet microchannels being connected to and in fluid communication with said fluid outlet hole 3.1 , o a nanochannel layout comprising a plurality of nanochannels, wherein each nanochannel is directly connected to and in fluid communication with said plurality of inlet and outlet microchannels, said plurality of nanochannels providing the sole fluid connection between said inlet microchannel and said outlet microchannel.

[0088] Furthermore, within the microfluidic and nanofluidic chip device according to the prior art the microchannel layout and the nanochannel layout are alternatively stacked in a manner that said inlet microchannel is adjacent to said outlet microchannel.

[0089] Example 2.- Measure of the pressure and flow rate that can be applied within the microfluidic and nanofluidic chip device according to the prior art in an operational mode.

[0090] FIG. 3B shows a time evolution of the pressure and of the flow rate of the fluid (or solution) within the microfluidic and nanofluidic chip device according to the prior art described in the Example 1 when said microfluidic and nanofluidic chip device is in an operational mode. The pressure and flow rate increase until a critical threshold of pressure of around 47.5 bar wherein a sudden drop of pressure is observed indicating a breakage of the chip device. Example 3.- Microfluidic and nanofluidic chip device according to the present invention.

[0091] Figure 4 shows in FIG. 4A a schematic longitudinal section of the first embodiment of the microfluidic and nanofluidic chip device according to the present invention (see also FIG. 1 ).

[0092] Example 4.- Measure of the pressure and flow rate that can be applied within the microfluidic and nanofluidic chip device according to the present invention.

[0093] FIG. 4B shows a time evolution of the pressure and of the flow rate of the fluid (or solution) within the microfluidic and nanofluidic chip device according to the first embodiment of the present invention when said microfluidic and nanofluidic chip device is in an operational mode. As can be seen, the pressure can increase within the chip device until a pressure of 90 bar without any problem. This shows a much stronger higher pressure endurance of the microfluidic and nanofluidic chip device according to the present invention as compared to the microfluidic and nanofluidic chip device according to the prior art. In addition, since the flow rate within the microfluidic and nanofluidic chip device according to the present invention can increase up to 10 times (as compared to the flow rate within the microfluidic and nanofluidic chip device according to the prior art), it allows having a microfluidic and nanofluidic chip device with scalable properties, rendering it convenient for GMP applications

[0094] In addition, the presence of the inlet (and also the outlet chamber) in the microfluidic and nanofluidic chip device according to the first embodiment of the present invention allows improving the distribution of the pressure within the chip device. This permits to increase the pressure in operational model while reducing the risk of breakage of the device.

[0095] Example 5.- Measurement of the loading efficiency of miRNA into Extracellular Vesicles (EVs) from HEK cells using the method and device of the present invention.

[0096] The inventors loaded 70 mL of a solution containing EVs from HEK cells, at a concentration of 1-5E1 1 EVs / mL, into the microfluidic and nanofluidic chip device described in the present invention (see Figure 5, EX5). The solution flowrate was set to approximately 1000 L / min, and the miRNA cargo (miRl 40) concentration was around 10 pM. The method of the present invention was applied, and the level of loaded miRl 40 was measured by RT-qPCR in the solution after it passed through the microfluidic and nanofluidic chip device. Total RNA is extracted from a defined volume of the EV preparation after removal of extra-vesicular nucleic acids, including non-encapsulated or non-vesicle- associated miRNA, in order to specifically quantify vesicle-associated miRNA (such as miR-140). An internal extraction control is added to each sample prior to RNA extraction, enabling monitoring of extraction and RT-qPCR performance on a per-sample basis. In addition, the overall RNA extraction efficiency is evaluated for each experimental run. miRNA such as for example miR-140 is quantified by RT-qPCR using a calibration curve generated with known amounts of synthetic miR-140, allowing conversion of Ct values into an absolute number of miR-140 molecules present in the analysed sample volume. In parallel, the concentration of EVs in the same preparation is determined using an independent particle counting method.

[0097] The average number of miR-140 molecules per EV is then calculated by dividing the total number of miR-140 molecules measured in the sample by the total number of EV particles in the corresponding volume.

[0098] The average loading efficiency reached 0.05 copies of miR-140 per EV. This level is comparable to the most abundant miRNAs naturally present in EVs from Wharton’s Jelly cells or from HEK cells. Figure 5 shows the average copy number of the most represented miRNAs in these EVs, from miR-1 to miR-21. With a loading efficiency of 0.05 copies of miR-140 per EV (EX5, Figure 5), the amount loaded using the present invention falls within the same range as these naturally abundant miRNAs.

[0099] Comparative Example 1.- Measurement of miRNA loading efficiency using a method and device from the prior Art (see for example EP401 1494).

[0100] The conditions of Example 5 were replicated using a method and microfluidic / nanofluidic chip device according to the prior art (e.g., EP401 1494) (see Figure 5, CE1 ). In this case, the flowrate was set at 501 L / min, and the maximum volume of the loading solution that could be processed was limited to 35 mL. Contrary to Example 5, the loading efficiency was significantly lower, with only 0.01 copies of miR140 loaded per EV.

[0101] The method of the present invention therefore yields a five-fold increase in loading efficiency compared with the prior art. It also allows the processing of double the volume and operates at double the flowrate used in the comparative example.

[0102] Example 6.- Measurement of the biological potency and stress inhibition of EVs loaded with miR-140

[0103] The inventors used the loaded EVs with miR140 obtained from Example 5 and from the Comparative Example 1 to measure the biological potency in vitro. It appears that EVs produced in Example 5 need four times fewer particles than the EVs produced in the Comparative Example 1 to achieve the same antiinflammatory effect as measured by the percentage of inhibition of IL-6 production compared to LPS induced reference level (IC50: 5.59E+08 EV / ml versus 2.25E+09 EV / ml, respectively).

[0104] In addition, the method for loading EVs according to the present invention or the microfluidic and nanofluidic chip device for loading EVs according to the present invention loads the molecule of interest (here miR-140) more effectively into EVs, which boosts their anti-inflammatory activity. Thus, the results indicate that increased loading efficiency strongly correlates with increased potency.

[0105] Figure 6 shows the dose response curves for EVs loaded with miR-140 from Example 5 and Comparative Example 1. EVs produced using the present invention display a steeper response curve, indicating that their anti-inflammatory effect increases more efficiently with higher EV concentrations. In contrast, the EVs produced using the prior art method show a flatter profile, consistent with reduced potency across the concentration range. These results confirm that the method and device described in this invention generate EVs with superior biological activity and a stronger dose dependent response.

[0106] It is understood that the present invention is in no way limited to the embodiments described above and that many modifications can be made thereto without departing from the scope of the appended claims.

Claims

24 CLAIMS1 . A method for loading biological particles with a molecule of interest comprising the steps of: selecting a microfluidic and nanofluidic chip device (1 ) comprising a fluid inlet (2.1 ) and a fluid outlet (3.1 ), one or several (identical) inlet microchannels (4.1 ) and outlet microchannels(4.2) having a constant section with its (their) smallest dimension in the micrometer range, and a plurality of identical nanochannels (4.3) having a constant section with their smallest dimension in the nanometer range, wherein the nanochannels (4.3) provide the sole fluid connection between said inlet microchannel (4.1 ) and said outlet microchannel (4.2), mixing the said biological particles with a solution comprising the said molecule of interest, applying the mixed biological particles solution with the said molecule of interest to the fluid inlet (2.1 ) of the said microfluidic and nanofluidic chip device (1 ), applying a pressure to the said microfluidic and nanofluidic chip device (1 ) forcing the biological particles and the molecule of interest to pass through a inlet microchannel (4.1 ), then to a nanochannel (4.3) so that pressure on the biological particle and / or physical forces cause the molecule of interest to enter the said biological particle and, then through an outlet microchannel(4.2), wherein at least 95% v / v, more preferably at least 98% v / v or even more preferably 99% v / v, of the mixed biological particles solution with the said molecule of interest entering through said fluid inlet is passing through said fluid outlet, recovering the said loaded biological particles.

2. The method of claim 1 where more than 108biological particles per milliliter are mixed with the solution comprising the molecule of interest.

3. The method of claim 1 or 2, wherein the flow velocity inside the microfluidic and nanofluidic chip device (1 ) is equal or higher than 0.01 meter per second, and is preferably comprised between 0.01 and 50 meter per second.

4. The method according to any of the preceding claims, wherein the molecule of interest is an oligonucleotide or a polynucleotide.

5. The method of claim 4, wherein the oligonucleotide is at a concentration between 1 nM and 1 mM.

6. The method of claim 4 or 5, wherein the oligonucleotide is a miRNA and / or a circular RNA.

7. The method according to any one of the preceding claims 4 to 6, comprising a final step of treating the loaded biological particles with a solution comprising a nuclease so as to remove non-internalized nucleic acid molecules.

8. The method according to any of the preceding claims, further comprising the step of polishing the composition comprising the loaded biological particles by filtration and / or centrifugation.

9. The method according to any of the preceding claims, wherein the flow rate within the microfluidic and nanofluidic chip device (1 ) is higher than 100 l / minute.

10. The method according to any of the preceding claims, wherein the section of the nanochannels (4.3) has one dimension comprised between 0.2 and 5 times the diameter of the biological particle.1 1 . The method according to any of the preceding claims, wherein the biological particle is surrounded by a bilayer essentially consisting of phospholipids.

12. The method according to any of the preceding claims, wherein the biological particle is an extracellular vesicle.

13. The method according to any of the preceding claims, wherein the biological particle has a diameter or equivalent diameter comprised between 30 and 2000 nm, preferably between 50 and 1000 nm, more preferably between 100 and 500 nm.

14. The method according to any one of the preceding claims, wherein the pressure inside the microfluidic and nanofluidic chip device (1 ) is set between 1 and 250 bar, preferably between 1 and 100 bars, more preferably between 5 and 50 bar, even more preferably between 10 and 30 bar.

15. A microfluidic and nanofluidic chip device ( 1 ) comprising: at least one fluid inlet (2) comprising at least one fluid inlet hole (2.1 ), at least one fluid outlet (3) comprising: o at least one fluid outlet hole (3.1 ), and o at least one fluid outlet chamber (3.2) being delimited by a bottom wall, an upper wall, and at least a side wall connecting said bottom wall to said16 upper wall in a sealed manner, said fluid outlet hole being located within said bottom wall and / or said upper wall, at least one fluid module (4) comprising: o a microchannel layout comprising at least one inlet microchannel (4.1 ) having a constant section with its smallest dimension in the micrometer range, said inlet microchannel (4.1 ) being connected to and in fluid communication with said fluid inlet (2), and at least one outlet microchannel (4.2) having a constant section with its smallest dimension in the micrometer range, said outlet microchannel (4.2) being connected to and in fluid communication with said fluid outlet chamber (3.2), o a nanochannel layout comprising a plurality of nanochannels (4.3), wherein each nanochannel (4.3) is directly connected to and in fluid communication with said inlet microchannel (4.1 ) and with said outlet microchannel (4.2), said plurality of nanochannels (4.3) providing the sole fluid connections between said inlet microchannel (4.2) and said outlet microchannel (4.2), each nanochannel (4.3) having a constant section with its smallest dimension in the nanometer range, wherein said at least one inlet microchannel (4.1 ) and said at least one outlet microchannel (4.2) are alternatively stacked in a manner that said inlet microchannel is adjacent to said outlet microchannel, and wherein each nanochannel (4.3) of the plurality of nanochannels (4.3) having a first longitudinal end (4.31 ) and a second longitudinal end (4.32), said ends (4.31 , 4.32) being opposite to each other along a longitudinal axis (L2) of each nanochannel, and wherein said ends (4.31 , 4.32) are directly connected to and in fluid communication either with said inlet microchannel(4.1 ) and / or with said outlet microchannel (4.2), and wherein said fluid outlet chamber(3.2) comprises at least one support column (3.3) connecting said bottom wall to said upper wall, said support column (3.3) being arranged to reinforce the structure of said fluid outlet chamber (3.2) by maintaining a predetermined distance between said bottom wall and said upper wall.

16. The microfluidic and nanofluidic chip device ( 1 ) according to claim 15, wherein said fluid inlet (2) further comprises at least one fluid inlet chamber (2.2) being delimited by a bottom wall, an upper wall, and at least a side wall connecting said bottom wall to said upper wall in a sealed manner, said fluid inlet hole (2.1 ) being located within said bottom wall and / or said upper wall, wherein said fluid inlet chamber(2.2) comprising at least one support column (2.3) connecting said bottom wall to said27 upper wall, said support column (2.3) being arranged to reinforce the structure of said fluid inlet chamber (2.2) by maintaining a predetermined distance between said bottom wall and said upper wall.

17. The microfluidic and nanofluidic chip device ( 1 ) according to claim 15 or claim 16, wherein(i) the fluid outlet chamber (3.2) having a first outlet chamber part(3.4) and a second outlet chamber part (3.5) and having a flared shape widening along a longitudinal axis (LI ) of said fluid outlet chamber (3.2) from a first end to a second end of said fluid outlet chamber (3.2), wherein said outlet hole (3.1 ) is located within said first outlet chamber part (3.4) of said fluid outlet chamber (3.2), said first outlet chamber part (3.4) being located nearer said first end of said fluid outlet chamber (3.2) than said second outlet chamber part (3.5), while said outlet microchannel (4.2) being connected to and in fluid communication with said second outlet chamber part(3.5), said second outlet chamber part (3.5) being located nearer said second end of said fluid outlet chamber (3.2) than said first outlet chamber part (3.4), and / or(ii) the fluid inlet chamber (2.2) having a first inlet chamber part (2.4) and a second inlet chamber part (2.5) and having a flared shape widening along a longitudinal axis (LI ) of said fluid inlet chamber(2.2) from a first end to a second end of said fluid inlet chamber(2.2), wherein said inlet hole (2.1 ) is located within said first inlet chamber part (2.4) of said fluid inlet chamber (2.2), said first inlet chamber part (2.4) being located nearer said first end of said fluid inlet chamber (2.2) than said second inlet chamber part (2.5), while said inlet microchannel (4.1 ) being connected to and in fluid communication with said second inlet chamber part (2.5), said second inlet chamber part (2.5) being located nearer said second end of said fluid inlet chamber (2.2) than said first inlet chamber part (2.4).

18. The microfluidic and nanofluidic chip device according to any of the claims 15 to 17, wherein said fluid module (4) comprising said nanochannel layout comprising a plurality of nanochannels (4.3) having each a longitudinal axis (L2) substantially parallel to each other, said nanochannel layout being located between28 an inlet microchannel layout comprising a plurality of inlet microchannels(4.1 ) and an outlet microchannel layout comprising a plurality of outlet microchannels (4.2), said plurality of inlet microchannels (4.1 ) and said plurality of outlet microchannels (4.2) having each a longitudinal axis (LI ) substantially parallel to each other and oriented substantially perpendicular to said longitudinal axis (L2) of said plurality of nanochannels.

19. The microfluidic and nanofluidic chip device ( 1 ) according to claim 18, wherein each nanochannel (4.3) of the plurality of nanochannels (4.3) having a first longitudinal end (4.31 ) and a second longitudinal end (4.32), said ends (4.31 , 4.32) being opposite to each other along the longitudinal axis (L2) of each nanochannel, and wherein said first longitudinal end (4.31 ) of each nanochannel (4.3) is directly connected to and in fluid communication with the first inlet microchannel (4.1 ) of the plurality of inlet microchannels (4.1 ) or the first outlet microchannel (4.2) of the plurality of outlet microchannels (4.2) while said second longitudinal end (4.32) of each nanochannel (4.3) is directly connected to and in fluid communication with the last inlet microchannel (4.1 ) of the plurality of inlet microchannel (4.1 ) or the last outlet microchannel (4.2) of the plurality of outlet microchannels (4.2).

20. The microfluidic and nanofluidic chip device according to any of the claims 15 to 1 , wherein each nanochannels (4.3) of said plurality of nanochannels(4.3) are substantially parallel to each other and wherein two adjacent nanochannels(4.3) of the plurality of nanochannels (4.3) are located at a distance comprised between 10 pm and 500 pm from each other.

21. The microfluidic and nanofluidic chip device (1 ) according to any of the claims 15 to 20, wherein said at least one inlet microchannel (4.1 ) having a constant section with its smallest dimension comprised between 1 pm and 1000 pm and / or wherein said at least one outlet microchannel (4.2) having a constant section with its smallest dimension comprised between 1 pm and 1000 pm.

22. The microfluidic and nanofluidic chip device (1 ) according to any of the claims 15 to 21 , wherein each nanochannel (4.3) of the plurality of nanochannels(4.3) have a constant section with its smallest dimension comprised between 20% and 1000% of the smallest dimension of a section of an extracellular vesicle (smallest dimension of the constant section of the nanochannel / smallest dimension of a section of an extracellular vesicle passing through said nanochannel).

23. The microfluidic and nanofluidic chip device (1 ) according to any of the claims 15 to 22, wherein said fluid module (4) having a length in said longitudinal29 axis (LI ) comprised between 15 mm and 80 mm and a width in said longitudinal axis (L2) comprised between 10 mm and 30 mm.

24. An assembly compatible with scalable Good Manufacturing Practice (GMP) production comprising multiple microfluidic and nanofluidic chip devices according to any of the claims 15 to 23, wherein the chip devices are arranged in parallel to each other, and wherein the assembly further comprises: a common inlet distribution system configured to supply each chip device with fluid under homogeneous and controlled flow conditions, the common inlet distribution system being connected to each fluid inlet of each microfluidic and nanofluidic chip device, a common or individually addressable outlet collection system configured to retrieve processed fluids from each chip device, the common or individually addressable outlet collection system being connected to each fluid outlet of each microfluidic and nanofluidic chip device, optionally, a supporting frame or module configured to maintain the relative positioning of the chip devices, such that the parallel arrangement of the chip devices enables linear upscaling of processing throughput while preserving the individual fluidic performance of each microfluidic and nanofluidic chip device.

25. A method of fabricating said microfluidic and nanofluidic chip device ( 1 ) according to any of the claims 15 to 23, comprising:- fabricating a plurality of nanochannels in a first substrate to obtain a nanochannel layout wherein each nanochannel (4.3) having a constant section with its smallest dimension in the nanometer range;- fabricating an fluid inlet (2) and an fluid output (3) in a second substrate,- fabricating at least one inlet microchannel (4.1 ) and at least one outlet microchannel (4.2) in said second substrate wherein said fluid inlet (2) is directly connected and in fluid communication with said at least one inlet microchannel (4.1 ) and wherein said fluid output (3) is directly connected and in fluid communication with said at least one outlet microchannel (4.2), wherein said at least one inlet microchannel (4.1 ) and said at least one outlet microchannel (4.2) are alternatively arranged at intervals to form a microchannel layout,- alternately stacking and bonding at least one nanochannel layout and at least one microchannel layout to obtain the micro- and nano-fluidic chip, wherein the at least30 one inlet microchannel (4.1 ) and the at least one outlet microchannel (4.2) are fluidly connected through said plurality of nanochannels (4.3).

26. Use of the microfluidic and nanofluidic chip device (1 ) according to any of the claims 15 to 23 or use of the assembly according to claim 24, to perform mechanoporation of biological particles surrounded by a lipid bilayer in pharmaceutical, GMP conditions.

27. A method for loading biological particles with a molecule of interest by implementing the microfluidic and nanofluidic chip device according to any of the claims 15 to 23, said method comprising the steps of: selecting said microfluidic and nanofluidic chip device according to the present invention, mixing the said biological particles with a solution comprising the said molecule of interest, applying the mixed biological particles solution with the said molecule of interest to the fluid inlet of the said microfluidic and nanofluidic chip device, applying a pressure to the said microfluidic and nanofluidic chip device forcing the biological particles and the molecule of interest to pass through said microchannel layout comprising at least one inlet microchannel, then to said nanochannel layout comprising a plurality of nanochannels so that pressure on the biological particle and / or physical forces cause the molecule of interest to enter the said biological particle and then through said at least one outlet microchannel in order to reach said fluid outlet,28. A pharmaceutical composition comprising biological particles, preferably extracellular vesicles, loaded with at least one exogenous nucleic acid molecule, preferably an exogenous microRNA, and wherein the biological particles are loaded at an average level of at least 0.03, preferably at least 0.04 or even preferably at least 0.05 exogenous nucleic acid molecule per biological particle, and, preferably, wherein the biological particles loaded with the exogenous nucleic acid molecules are obtainable by implementing the method according to claims 1 to 14 or by implementing the device according to claims 15 to 23 or by implementing the method according to claim 27.

29. The pharmaceutical composition according to claim 28, wherein the biological particles are extracellular vesicles, preferably extracellular vesicles from immortalized mesenchymal stem cells such as Wharton’s Jelly cells, or from HEK cells.3130. The pharmaceutical composition according to claim 28 or claim 29, comprising at least 1010extracellular vesicles loaded at an average level of at least 0.03 exogenous nucleic acid molecule per biological particle (extracellular vesicle), wherein the exogenous nucleic acid are measured by RT-qPCR and wherein the exogenous nucleic acid molecules are a miRNA and / or a circular RNA, preferably the miRNA is miR- 140, miR-21 , and / or miR-146.

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