FR2839454A1 - DEVICE FOR MICRO-INJECTION OF LIQUID IN A CONFINED MEDIUM - Google Patents

Abstract

The present invention relates to a device for the micro-injection of liquid in a confined environment comprising at least: - a syringe (16) with piston (19) carrying an injection cannula (23) of small diameter, - drive means of the piston for injection, - control means for the piston drive means, the drive means being of the pneumatic type controlled by a movable member that can be actuated by an operator, the injection volumes being less than approximately 800 l.

Description

novirhabdovirus in fish.

  The present invention relates to a device for micro-injection

  of liquid in a confined medium in vivo.

  In particular, the present invention relates to a device making it possible to perform subretinal injections of small volumes controlled and automated by transvitreenne flies in an eyeball,

  including a large eyeball.

  It is known to carry out subretinal injections for the gene transfer or the transfer of cells in the refine of an animal of

  large size, in the field of ocular gene therapy.

  In a stereotaxic method, for the automatic dosing of the liquid to be injected, Russel et al. (A Manual, Remotely Actuated Injector For SubRetinal Gene Therapy in Non-Human Primates. IOVS March 1S, 2001, vol. 42, N 4) describe the use of an electric stepper motor that pushes the plunger of a syringe 100 pI Hamilton type connected to a glass micropipette having an end of 10 µm in diameter previously positioned in the subretinal space of monkeys, with a micromanipulator. The injected volume is controlled by the electric stepper motor. This method does not allow the surgeon much

  maneuverability of the cannula which limits the choice of injection site on the retina.

  Furthermore, Fong-Qi Liang et al. (Intraocular Delivery of Recombinant Virus, Methods in Molecular Medicine, vol. 47: Vision Research Protocols, pp. 125-139) describe transvitinal intraocular injections of recombinant virus into dog eyes. According to this technique, a gauges needle is introduced through a sclerotomic incision through the vitreous body. The cannula is connected to a rubber plunger syringe containing the viral liquid to be injected, this plunger syringe being connected to a 1 ml Hamilton type syringe filled with saline liquid. The surgeon directs the needle of the cannula into the target area, and an assistant holds the annex syringe filled with saline fluid. When the needle reaches the retina, on the surgeon's order, the assistant suddenly injects a volume; predefined fluid at the adjoining syringe of saline liquid, which

  gives an injection of purified virus into the subretinal space.

  The surgeon has great freedom of maneuver to introduce and

  position the cannula on a precise site of the retina.

  However, the pushing of the liquid is manual and requires the

  presence of an assistant. The subretinal injection is not reproducible.

  For transvitreenne injection of viral solutions, practitioners are faced with problems of low volumes precise to deliver, without loss

  virus solution and precision of the injection site itself.

  Those skilled in the art confronted with the problem of precise injection of micro-quantities are tended to use, for an automated injection, non-compressible fluids such as water or saline liquid or else a

  electric control, for example an electric stepper motor.

  With an electric stepping motor, mechanical connections, or non-compressible fluids, phenomena of dead strokes and wind transmission clearance induced by the mode of pressure transmission and

  themselves induce imprecision on the quantity of liquid delivered.

  In the present invention, surprisingly, a compressible fluid is used for the precise injection of micro-quantities. The compression of the fluid is used as a buffer which absorbs the play, the vibrations and the micro-shocks and the surgeon can actuate the pedal according to what he volts. Thus the automatic system allows the damping of undesirable phenomena induced by the systems

known injection.

  In a particular embodiment, the pedal allows the setting in pressure of the pressure and its stopping. The pressure is preset to a given value of the pressure of the liquid to be injected. The surgeon therefore has direct visual control of the size of the cavity created by the detachment, also called "bleb" when he injects, and can stop the injection as soon as the size of the

  desired retinal detachment is achieved.

  It is however possible to provide a device where the pedal would allow not only the implementation of the pressure but also the adjustment thereof. Indeed, when using small diameters of injection cannula, a pressure drop phenomenon is created which itself creates a resistant force retransmitted to the control pedal. The surgeon can feel the force feedback on the control, which allows him to modulate

['effort applied to the pedal.

  Thus, the system described in the present inversion makes it possible to combine the two advantages. On the one hand, the part held by the surgeon during the injection is handy, and the injection site, chosen for example on the refine, is therefore easily reached. On the other hand, the system is automated

  and is however completely controlled by the surgeon himself.

  Thus, the device according to the invention for the micro-injection of liquid in a confined medium comprises: - a piston syringe carrying an injection cannula of small diameter, - means for driving the piston for [injection, - control means for the piston drive means, the drive means being of the pneumatic type controlled by a movable member actuable by an operator,

  injection volumes being less than about 800, ul.

  By "cannula" is meant here a sleeve device adaptable to the distal end of a syringe and which comprises, at its distal end, a needle of sickle diameter. The "cannulas" are defined by the dimension

  of this falble diameter needle.

  The connection means between the syringe and the wind pressure generator preferably flexible and thus allow good manipulation of the syringe by the operator, unlike any other

mechanical system.

  According to the invention, the control fluid is a compressible gas which induces a direct action on the piston: the implementation of the device

  does not lead to the creation of games and the inaccuracies are absorbed.

  The surgeon performs a visual control of the injection and can deliver the pneumatic pressure by acting on the pedal, without having to use an assistant. Problems of volume control and loss of solution

viral wind thus resolved.

  We describe here a method of applying a therapeutic substance in a confined space, in particular inside the body of a patient and more particularly in the subretinal space, the method comprising the steps consisting in providing an element tubular elongate having a proximal end and a distal end, said tubular member comprising a cannula at its distal end and proximal end being connected to a syringe with a movable plunger filled with a therapeutic substance to be injected, and to connect said syringe to a system compressed air, the syringe and the system being adapted to communicate pneumatically by flexible connection means, to provide means for controlling the pressure by means of a movable member operable by an operator, for positioning the distal end of [ tubular element on the tissue to be treated, and to inject the therapeutic substance and visually control the

volume injects.

  In the application to subretinal injections by transvitreen flies, it is possible to inject at different places on the retina, from

preferably around the taste bud.

  Figure 1 represents in fa, schematically a device according to the

present invention.

  In Figure 2, the syringe device is more particularly shown. Figure 3 represents a cannula of 44 gauges usable according to

[invention, front view.

  In FIG. 4 are represented retinal images showing the expression of GFP (Green Fluorescent Protein of the jellyfish Aequorea l s victoria expressed in the refine after sub-retinal injection of vectors

  recombinants carry the GFP gene in dogs.

  According to the invention, preferably the injection volumes are between approximately 20 and approximately 600 μl, more preferably between approximately 50 and approximately 250 μl. Preferably, in the device according to the invention, the volume of the

  syringe reservoir is less than about 1,000, ul.

  Preferably, according to the invention, for subretinal injections by transvitreenne flies, the size of the cannula is preferably understood

  between 28 and 44 gauges and in a particular mode of the order of 44 gauges.

  For the injection of cells a cannula of 28 to 33 gauges will be preferred and for the injection of vectors a cannula of 44 gauges will be preferred. In the device according to the invention, the drive means comprise a pressurized gas reservoir connected to the piston syringe, by a control unit comprising an adjustable regulator with electronic control. Preferably, according to the invention, the gas tank contains air

  dry clean, including medical grade air.

  Preferably, according to the invention, the confined space is the retinal space, in particular the confined space located between the layer of

  photoreceptors and the retinal pigment epithelium.

  The microinjection described here may however have other applications, in particular in gene therapy or cell therapy. In these techniques, it is interesting that a single operator injecting small quantities of liquid in a confined space can operate a visual control of the

  injection site and control the amount injected.

  Several injection sites in the eye, of vectors or cells, can be targeted: subretinal by transvitreenne fly, intravitreen (in

  the window) and intracamerularly (in the anterior chamber).

  An ocular, gene or cell therapy drug, injected according to any of the above routes, could be composed of the preconditioned device with a certain volume of vectors or cells. Gene therapy means a gene therapy carried out using viral, non-viral vectors or nut II DNA. It can be any vector carrying a therapeutic or non-therapeutic gene, whatever its tail.

  origin. Diagnostic applications are also possible.

  For the subretinal injection by transvitreenne fly, the length of the needle carried by the cannula is adapted to the size of the eye. In other applications, it is adapted to the approach of the confined space to be reached. A particular embodiment of the piston drive means and control means for these drive means is

described in Figures 1 and 2.

  Thus, the pneumatic type drive means can be constituted by a viscous fluid extraction and injection system known as DORC VFINFE which implements an electronic pressure reducer and a Venturi suction system feeds with a

  external pressure of 5 to 6 bars, for example.

  Such systems are usually used for large volume intraocular injections and extractions, namely several milliliters,

  usually 10 to 20 ml which employ viscous liquid.

  With regard to gene therapy, the volumes injected are for example of the order of 50 to 250 μl, and this technique of intraocular injection requires the injection of small volumes, without loss.

  product and preferably university, it is at least partially automated.

  The pressure generator is according to the invention connected to the plunger of a micro-quantity injection syringe by connection means

gas and liquid tight.

  As shown in FIG. 1, these pneumatic drive means comprise a control unit 1 comprising a holder 2 connected to a gas reservoir 3, for example a bottle or the network of the hospital. A pedal 4, for example on foot, controls the injection of gas in the direction of the arrow 5, in the direction of the piston 9 of the syringe 10. The connection and pipe 7, 8 means for the gas from the reservoir 3 ['central unit 1 and [' unit 1 to the piston 9 of the syringe 10, as well as the

  connection means 6 of line 8 to the syringe 10 airtight.

  The means 8 for driving the wind gas at least partially flexible for

  allow manipulation of the syringe 10 by the operator.

  In addition, when the pedal is released, the system returns the

pressure at zero in the circuit.

  In the embodiment of the injection device 10 represented in FIG. 2 and in FIG. 3 for the detail of the cannula 17, the connection tube 18 to the pressure generator 1 comprises a connector 11 to the generator. This connector is itself connected by a double luer-iock male / male connector 12, connected to a female / female connector 13 itself connected to another male / male connector 14 partially engaged in the body 15 of a syringe 16 of 1 ml on which is fitted a cannula 17: the sleeve 24 carries a needle 23 of 44 gauges. A clip-on junction piece 21 is provided between the male / male connector 14 and the body of the syringe 15. Furthermore, preferably, a cap 20 for locking the cannula is provided, in particular so that the cannula does not become detached from the body of the syringe under the effect of the pressure which comes through line 18 into the body 15 of the syringe 16 and pushes the rubber plunger 19 which closes the

  volume of reservoir 22 of the syringe.

  Thus, in the particular embodiment described, the elements making up the wind plunger syringe device in particular: - a double male-male luer-lock connection (Vycon): 12 - female / female connection (AILON Total plus vitreoctomy pack, Alcon surgical): 13 - mfile / male connection (AILON Total plus vitreoctomy pack, Alcon surgical): 14 - a metal clip: 21 - 1 ml syringe: 16 - a 17 needle cannula 23 of 44 gauges carried by the sleeve 24 adapted to the syringe locking cap of the cannula (piece of the protective cap of a catheter with Becton-Dickinson fins): 20 The 44 gauges cannula represented in Figure 3 has for commercial references: at DORC International BV ("INJEC.NMLD 0.1 TIP STERILE"). In the particular embodiment, the material of the needle coming into contact with the refine is polyimide. As shown in FIG. 3, the needle 23 carried by the sleeve 24 of the cannula 17 has a

0.1 mm outside diameter.

  EXAMPLE After filling the 1 ml syringe with the solution to be injected, the operator removes the plunger handle from the syringe while keeping the syringe on the side of the rubber plunger. The operator then adapts the female / male and male connections to the clamp and fixes them there using the metal connecting piece. The operator then fixes the male / male double luer-lock connector and connects the female connector

  of the pressure generator controlled on the double luer-lock male connector.

  Once the cannula is attached to the end of the syringe, it is blocked by

['insertion of the cap.

  After having started the pressure generator, and made the choice of the adapted pressure, the operator performs a sclerotomy on the eye, he inserts the cannula through the glass until the refine and activates the injection in infancy, cant

  The foot switch, under its own visual control.

  Results Using this new device, subretinal injections of recombinant Adeno-Associated Virus (MV) virus and carrying the gene

  gfp marker (rMVgfp) were performed in the Beagle dog eye.

  Sub-retinal injections of 50 to 100 µl of viral solution led to retinal detachments of 0.6 and 1.3 cm2 respectively. Efficient gene transfer was obtained with 90-100% of photoreceptors

  and pigmented epithelium cells transduced to the target area.

  As shown in Figure 4, images of the bottom of the eye

  show the expression of GFP at different times after injection of rMV-

  2.CMVgfp in two dogs, BD1 (A to D) and BD2 (E to H).

  The morphology of the refine was checked throughout the experiment and no anomalies were detected. (Has been). injection point. Pigmented scars due to two attempts at negative injection (obstruction of the cannula). The fluorescent images show the expression of GFP at 6 days (B. F), 30 days (C, G) and 90 days post-injection.

Claims (8)

  1. Device for micro-injection of liquid in a confined medium comprising at least: - a syringe (16) with piston (9, 19) carrying an injection cannula (23) of small diameter, - - drive means piston for injection, control means for the piston drive means; the drive means being of the pneumatic type controlled by a movable member actuable by an operator,   1 injection volumes being less than about 800 pI.   2. Device according to claim 1, characterized in that the volumes - -   wind injection between 20 and 600 pI.   3. Device according to claim 1 or 2, characterized in that the   wind injection volumes between 50 and 250 IJI.   4. Device according to one of claims 1 to 3, characterized in that   the size of the cannula (23) is less than 28 to 44 gauges.   5. Device according to one of claims 1 to 4, characterized in that   the volume of the reservoir (22) of the syringe is. less than about 1000 pI .:   6. Device according to one of claims 1 to 5, characterized in that   the drive means comprise a gas tank (3) under pressure connected to the syringe (16) with piston (9, 19) by a control unit   (1) comprising an adjustable regulator (2) with electronic control (4).   7. Device according to one of claims 1 to 6, characterized in that   the gas tank contains clean dry air, especially of medical quality.   8. Device according to one of claims 1 to 7, characterized in that   the length of the needle 23 carried by the cannula 17 is adapted to the size