Microfluidic and nanofluidic chip device

Abstract

Microfluidic and nanofluidic chip device and method for loading biological particles with a molecule of interest, said chip device comprising a fluid inlet, a fluid outlet, and a fluid module comprising at least a inlet microchannel, an outlet microchannel and a plurality of nanochannels, wherein the longitudinal ends of each nanochannels are directly connected to and in fluid communication either with a inlet microchannel (4.1) and / or with an outlet microchannel (4.2), 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

Description

Technical Field

[0001] The present invention relates to a microfluidic and nanofluidic chip device.

[0002] The present invention also relates to a method of fabricating the microfluidic and nanofluidic chip device according to the present invention.

[0003] 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.

[0004] 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.State of the art

[0005] 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.

[0006] 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.

[0007] 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 return 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.

[0008] 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.

[0009] Microfluidic chips have been used in cellular loading. In particular, EP4011494 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. EP4011494 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 EP4011494 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 micro-environment 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.

[0010] Consequently, 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.

[0011] 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

[0012] The present invention aims to overcome the drawbacks of the state of the art, in particular those described above.

[0013] 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.Summary of the invention

[0014] 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: ∘ at least one fluid outlet hole, and ∘ 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: ∘ 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, ∘ 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.

[0015] 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 an 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.

[0016] Other embodiments of the microfluidic and nanofluidic chip device according to the present invention are indicated in the appended claims.

[0017] The present invention also relates to a method of fabricating the microfluidic and nanofluidic chip device according to the present invention, comprising: 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; fabricating a fluid inlet and a fluid output in a second substrate, 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, 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.

[0018] Other embodiments of the method of fabricating the microfluidic and nanofluidic chip device according to the present invention are indicated in the appended claims.

[0019] 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.

[0020] Other embodiments of the use of the microfluidic and nanofluidic chip device according to the present invention are indicated in the appended claims.

[0021] The present invention also relates to a method for loading biological particles with a molecule of interest comprising the steps of: selecting a microfluidic and nanofluidic chip device comprising a fluid inlet and a 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.Detailed description of the invention:

[0026] 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. 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. Figure 3 shows a schematic representation of the microfluidic and nanofluidic chip device according to the prior art (EP4011494) together with a pressure profile in an operational mode within this microfluidic and nanofluidic chip device according to the prior art. 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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: ∘ at least one fluid outlet hole 3.1, and ∘ 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: ∘ 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, ∘ 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 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.

[0032] 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 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 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.

[0033] 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 L1 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 L1 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.

[0034] 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 L1 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 L1 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.

[0035] 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 L1 substantially parallel to each other and oriented substantially perpendicular to said longitudinal axis L2 of said plurality of nanochannels.

[0036] 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.

[0037] 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 µm and 500 µm 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 µm and 500 µm from each other.

[0038] Preferably, the at least one inlet microchannel 4.1 has a constant section with its smallest dimension comprised between 1 µm and 1000 µm, more preferably between 40 µm and 500 µm, and / or wherein said at least one outlet microchannel 4.2 having a constant section with its smallest dimension comprised between 1 µm and 1000 µm, more preferably between 40 µm and 500 µm.

[0039] 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).

[0040] 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).

[0041] Preferably, the fluid module 4 has a length in said longitudinal axis L1 comprised between 15 mm and 80 mm and a width in said longitudinal axis L2 comprised between 10 mm and 30 mm.

[0042] 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.

[0043] 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.

[0044] Preferably, in the method for loading biological particles with a molecule of interest according to the present invention, more than 10 8< biological particles per milliliter are mixed with the solution comprising the molecule of interest to form a mixed solution.

[0045] Preferably, the mixed solution comprises a phosphate-buffered saline (PBS), normal saline, Tris, Tris saline, or HEPES buffer.

[0046] 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.

[0047] Preferably, the flow rate within the microfluidic and nanofluidic chip device is equal or superior to 100 µl / minute. More preferably, the flow rate within the microfluidic and nanofluidic chip device is comprised between 100 and 2000 µl / minute, even more preferably between 500 and 2000 µl / minute.

[0048] 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 µm, more preferably equal to or smaller than 0,2 µm 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.

[0049] 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 1U / mL and 50U / mL, preferably between 10U / 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.

[0050] Preferably, the molecule of interest is an oligonucleotide or a polynucleotide.

[0051] Preferably, the oligonucleotide is at a concentration between 1 nM and 1 mM.

[0052] Preferably, the oligonucleotide is at least a miRNA and / or a circular RNA.

[0053] 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 non-internalized nucleic acid molecules.

[0054] 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.

[0055] Preferably, the section of the nanochannels has one dimension comprised between 0.2 and 5 times the diameter of the biological particle.

[0056] Preferably, the biological particle is surrounded by a bilayer essentially consisting of phospholipids.

[0057] Preferably, the biological particle is an extracellular vesicle.

[0058] 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.

[0059] 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.Examples.-

[0060] 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.

[0061] Figure 3 shows a schematic representation of the microfluidic and nanofluidic chip device according to the prior art (EP4011494) (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: ∘ 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, ∘ 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. 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.

[0062] 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.

[0063] 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.

[0064] Example 3.- Microfluidic and nanofluidic chip device according to the present invention.

[0065] 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).

[0066] 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.

[0067] 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

[0068] 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.

[0069] 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

1. 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: ∘ at least one fluid outlet hole (3.1), and ∘ 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: ∘ 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), ∘ 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.

2. The microfluidic and nanofluidic chip device (1) according to claim 1, 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 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.

3. The microfluidic and nanofluidic chip device (1) according to claim 1 or claim 2, 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 (L1) 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 (L1) 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).

4. The microfluidic and nanofluidic chip device according to any of the preceding claims, 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 between 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 (L1) substantially parallel to each other and oriented substantially perpendicular to said longitudinal axis (L2) of said plurality of nanochannels.

5. The microfluidic and nanofluidic chip device (1) according to claim 4, 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) .

6. The microfluidic and nanofluidic chip device according to any of the preceding claims, 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 µm and 500 µm from each other.

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

8. The microfluidic and nanofluidic chip device (1) according to any of the preceding claims, 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).

9. The microfluidic and nanofluidic chip device (1) according to any of the preceding claims, wherein said fluid module (4) having a length in said longitudinal axis (L1) comprised between 15 mm and 80 mm and a width in said longitudinal axis (L2) comprised between 10 mm and 30 mm.

10. A method of fabricating said microfluidic and nanofluidic chip device (1) according to any of the claims 1 to 9, 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 a fluid inlet (2) and a 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 least one inlet microchannel (4.1) and the at least one outlet microchannel (4.2) are fluidly connected through said plurality of nanochannels (4.3).

11. Use of the microfluidic and nanofluidic chip device (1) according to any of the claims 1 to 9, to perform mechanoporation of biological particles surrounded by a lipid bilayer in pharmaceutical, GMP conditions.

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