WO2022167536A2

WO2022167536A2 - Microfluidic process for the preparation of hydrogel-loaded liposome nanostructures

Info

Publication number: WO2022167536A2
Application number: PCT/EP2022/052605
Authority: WO
Inventors: Felicia ROFFO; Enza TORINO; Paolo Antonio Netti
Original assignee: Kyme Nanoimaging Srl
Priority date: 2021-02-05
Filing date: 2022-02-03
Publication date: 2022-08-11
Also published as: WO2022167536A3; IT202100002537A1

Abstract

A process for the preparation of self-assembled nanoparticles consisting of a lipid bilayer shell and an hydrogel core wherein active agents are entrapped by using a microfluidic device characterised in loading in the middle and sides channels of the microfluidic device a lipid stock solution, a solvent solution and a polymer solution, at specific concentrations and at a specific flow rate, to obtain the core-shell nanoparticles with desired features which can be used as contrast agents for multimodal imaging and in theranostic therapy.

Description

MICROFLUIDIC PROCESS FOR THE PREPARATION OF HYDROGEL-LOADED LIPOSOME NANOSTRUCTURES

Background of the invention

The present invention refers to the field of chemistry since it concerns the productions of nanoparticles to be used for diagnostic and therapeutic purposes. More in particular, the present invention refers to a process for the preparation of shell-core nanoparticles by means of microfluidic techniques, which can entrap active ingredients to be used as contrast agents for multimodal imaging and in theranostic therapy.

Prior art

Cheng, Z . , Thorek, D. L. J. & Tsourkas, A. Porous Polymersomes with Encapsulated Gd-labeled Dendrimers as Highly Efficient MRI Contrast Agents. Adv. Fund. Mater. 19, 3753-3759 (2009) synthesized a composite MR contrast platform, consisting of dendrimer conjugates encapsulated in porous polymersomes. The porous polymersomes, ~130 nm in diameter, were produced through the aqueous assembly of the polymers, polyethylene oxide-b- polybutadiene (PBdEO) , and polyethylene oxide-bpolycaprolactone (PEOCL) . Subsequent hydrolysis of the caprolactone (CL) block resulted in a highly permeable outer membrane. To prevent the leakage of small Gd-chelate through the pores, Gd was conjugated to PAMAM dendrimer via diethylenetriaminepentaacetic acid dianhydride (DTPA dianhydride) prior to encapsulation. The obtained nanoparticles exhibit improved permeability to water flux and a large capacity to store Gd-chelates within the aqueous lumen, resulting in enhanced longitudinal relaxivity. As a result of the slower rotational correlation time of Gd-labeled dendrimers, the porous outer membrane of the nanovesicle, and the high Gd payload, these functional nanoparticles were found to exhibit a relaxivity (Rl) of 292, 109 mM-ls-1 per particle. The polymersomes were also found to exhibit unique pharmacokinetics with a circulation half-life of >3.5 hrs and predominantly renal clearance. When injected into living subjects, the gradual destabilization of the polymersomes allow the Gd-labeled dendrimers to be predominantly cleared by the kidneys , while still maintaining a relatively long circulation time of >3 . 5 hrs . This could provide an important advantage over other long circulating MRI CAs , which generally require metabolism of the CA in the liver and exhibit prolonged retention in the body . In addition, with their work the authors showed also a way to prevent the leakage of encapsulated Gd through the membrane pores of the nanoconstructs .

Bui , T . et al . Novel Gd Nanoparticles Enhance Vascular Contrast for High-Resolution Magnetic Resonance Imaging . PLoS ONE 5 , ( 2010 ) discloses the production of lipid NPs containing phospholipids that express Gd-chelate or DTPA by incorporating DTPA-PE into the lipid core of the NPs and then adding Gd3+ to preformed NPs ( for binding to Gd3+ as Gd-DTPA-PE chelate ) . They also add 10-mole percentage of lipid conj ugated to mPEG-PE to lipid nanoparticles in order to increase the bound water on the lipid nanoparticle surface , thereby increasing the MRI contrast . In this case , the following nanoparticle system shows a higher longitudinal relaxivity ( 33-fold) than the current FDA approved Gd-chelated CAs . In addition, intravenous administration of these Gd-LNP at only 3% of the recommended clinical Gd dose produce MRI signal-to- noise ratios of greater than 300 times in all vasculatures .

Liao , Z . et al . Multifunctional Nanoparticles Composed of A Poly ( dl-lactide-coglycolide ) Core and A Paramagnetic Liposome Shell for Simultaneous Magnetic Resonance Imaging and Targeted Therapeutics . Adv. Fund . Mater . 21 , 1179-1186 ( 2011 ) discloses a core-shell NPs system composed of a PLGA core and a paramagnetic liposome shell for simultaneous MRI and targeted therapeutics . They encapsulate Dox within biocompatible and FDA-approved PLGA NPs , and DTPA-Gd is conj ugated to the amphiphilic octadecyl-quaternized lysine- modified chitosan ( OQLCS ) . The paramagnetic liposome shell is based on Gd-DTPA-conj ugated OQLCS (Gd-DTPA-OQLCS ) , folate- conj ugated OQLCS ( FA-OQLCS ) , and PEGylated OQLCS ( PEG-OQLCS ) . Briefly, the carboxyl groups of DTPA used as a chelating agent are combined with the amino groups of OQLCS . Then Gd is incorporated into the complex . As a result , the NPs show paramagnetic properties with an approximately 3-fold enhancement in the longitudinal relaxivity ( rl = 14 . 381 mM-ls-1 ) compared to the commercial Gd-DTPA complex and exceptional antitumor effects without systemic toxicity .

Gianolio , E . et al . Relaxometric investigations and MRI evaluation of a liposome-loaded pH-responsive gadolinium ( II I ) complex . Inorg . Chem . 51 , 7210-7217 ( 2012 ) reports the preparation of pH- responsive Gd-D03Asa-loaded liposomes which maintain the pH responsiveness of the unbound paramagnetic complex, and their relaxivities are markedly affected by the magnetic field strength, exhibiting a steep change in the relaxivity in the pH range 5 -7 . 5 . Moreover , they provide a ratiometric method for measurement of the pH based on a comparison of the relaxation effects at different magnetic fields , offering an alternative tool for accessing measurement of the pH without prior knowledge of the concentration of the paramagnetic agent .

Smith, C . E . et al . A polymeric fastener can easily functionalize liposome surfaces with gadolinium for enhanced magnetic resonance imaging . ACS Nano 7 , 9599- 9610 ( 2013 ) discloses a process to load Gd exclusively on a liposome surface using a polymeric fastener . The fastener, so named for its ability to physically link two functional components together, consisted of chitosan substituted with diethylenetriaminepentaacetic acid ( DTPA) to chelate gadolinium, as well as octadecyl chains to stabilize the modified chitosan on the liposome surface . The assembly strategy, mimicking the mechanisms by which viruses and proteins naturally anchor to a cell , provided greater T1 relaxivity than liposomes loaded with gadolinium in both the interior and outer leaflet . Liposomes were prepared by a film hydration method followed by sonication . The lipid film, formed as described above , was then hydrated with the aqueous mixture of gadolinium and DTPA-chitosan-g-C18 . The proposed process decouples particle assembly and functionalization and has considerable potential to enhance imaging quality while alleviating many of the difficulties associated with multifunctional particle fabrication . The average diameters of liposomes before and after one-hour incubation in serum- supplemented PBS were 4.3 ± 2 and 3.7 ± 2 pm respectively. Gadolinium loaded on the liposome modified by DTPA-chitosan-g-C18 significantly enhanced MR signal, compared to the liposome modified with DTPA-chitosan. At a given liposome concentration, R1 of the suspension was increased with DTPA-chitosan-g-C18. However, the molar relaxivity of immobilized gadolinium was nearly the same across samples. Therefore, authors interpreted that the enhancement of R1 attained with DTPAchitosan-g-C18 is due solely to the higher loading of gadolinium on the liposome surface, noting that 30% of the DTPA-chitosan was desorbed upon exposure to GdC13. Furthermore, the relaxivity was enhanced beyond that of the clinically used unconjugated DTPA-Gd complex with a molar relaxivity of 4.85 mM-ls-1. Authors also evaluated Gadolinium- coated liposomes in vivo using murine ischemia models to highlight the diagnostic capability of the system.

Cittadino, E. et al. In Vivo Magnetic Resonance Imaging Detection of Paramagnetic Liposomes Loaded with Amphiphilic Gadolinium ( III ) Complexes: Impact of Molecular Structure on Relaxivity and Excretion Efficiency, in (2013) reports the investigation on in vitro ( relaxometry) and in vivo (melanoma tumour model on mice) MRI performance of liposomes incorporating either LIPO-GdDOTA- (GAC12)2 (LIPO=liposome, GA=glutaric acid) or an amphiphilic monoamide Gd agent conjugated with C18 chains LIPOGdDOTAMA (C18 ) 2 as a reference. The liposomes were prepared using a thin-layer deposition/extrusion technique. The mean hydrodynamic size of the liposomes was found to be around 140 nm with a polydispersity index value lower than 0.2. Through the NMRD profiles, authors showed a marked relaxivity difference over the entire frequency range investigated, and their shape is rather similar and typical of macromolecular systems characterised by a reduced rotational tumbling rate. They distinguished: 1) a region of constant relaxivity at low fields (0.01-0.5 MHz) ; 2) a dispersion around 1- 3 MHz; 3) a peak centred about 20-30 MHz; and 4) a steep decrease of rl at higher fields. However, although for LIPO-GdDOTAMA (C18 ) 2 the rl peak (rl=11.4 mM-ls-1) is broad and centred at 30 MHz, for LIPO-GdDOTA (GAC12 ) 2 it is narrower and with a maximum at 20 MHz (rl=40.0 mM-ls-1) . As far as the in vivo studies, after about 7 h post-injection the contrast enhancement observed for the more efficient liposomes decreases rapidly and becomes lower than for LIPO-GdDOTAMA (C18 ) 2. The relaxometric data and the quantification of the Gd complexes in the organs indicated that: 1) the differences in the contrast enhancement can be attributed to the different rate of water exchange and rotational dynamics of the Gd complexes; 2) the rapid contrast decrease is caused by a faster clearance of GdDOTA (GAC12 ) 2 from the organs. The overall results highlighted clearly the superior relaxometric performance of the liposomes loaded with GdDOTA (GAC12 ) 2 relative to the liposome formulation based on the GdDOTAMA (C18 ) 2 complex. Therefore, GdDOTA (GAC12 ) 2 complex may represent a good candidate for the development of improved MRI protocols based on paramagnetically labelled lipidic nanoparticles .

Hossann, M. et al. Non-ionic Gd-based MRI contrast agents are optimal for encapsulation into phosphatidyldiglycerol-based thermosensitive liposomes. J. Control. Release Off. J. Control. Release Soc. 166, 22-29 (2013) investigated the formulations of 6 clinically approved CAs encapsulated into thermosensitive liposomes (TLs) and observed that Omniscan™ and Prohance® are the most promising candidates to be encapsulated into DPPG2-TSL. In particular, Prohance® allows the highest loading capability (256 mM) due to the lowest osmolality and yields the highest relaxivity. Omniscan™ is the only formulation that could be stored at 4 °C for weeks. The other CAs induce phospholipid hydrolysis, which results in unwanted CA leakage, and therefore reduce the shelf life of TSL. Nevertheless, Omniscan™ is associated with Nephrogenic Systemic Fibrosis (NSF) . The Human Serum Albumin (HSA) and Immunglobulin G (IgG) contribute to the increase of MRI signal at 30°C by increasing Pd. A high concentration of encapsulated CA is a prerequisite to achieve a sufficiently high Ari during heat triggered CA release combined with a low rl at 37°C. Hence, the optimal CA is characterized by a non-ionic structure and a low contribution to osmolality.

Cheng, Z. et al . Stabilized porous liposomes with encapsulated Gd- labeled dextran as a highly efficient MRI contrast agent. Chem. Commun. Camb. Engl. 50, 2502-2504 (2014) discloses a highly efficient MRI CA based on stabilized porous phospholipid liposomes with encapsulated Gd-labeled dextran. Unilamellar liposomes were prepared using the film hydration method. To prevent small Gd- chelates (i.e. Gd-DOTA) from leaking through the porous bilayer membranes, Gd-DOTA was attached to large molecular weight dextran prior to encapsulation (this attachment increased proton relaxivity of GdDOTA by slowing the molecular rotation) . In particular, 1 mL of Gd-DOTA-dextran (10 mg/mL) was added to the dried lipid film (2 mg of lipid) while non-entrapped Gd-DOTA- dextran was removed through repeated washing on centrifugal filter devices. The mean diameter of the liposomes was found to be about 100 nm. The highly porous membrane leads to a high relaxivity of the encapsulated Gd. Gd-DOTA-dextran encapsulated within the porous liposomes had an rl of 9.9 mM-ls-1, which was similar to the rl of Gd-DOTA-dextran (9.4 mM-ls-1) in bulk water. This indicates that Gd-DOTA-dextran encapsulated within the porous liposomes experiences a fast water exchange rate with surrounding bulk water. In contrast, Gd-DOTA-dextran encapsulated within the nonporous liposomes had an rl of only 4 mM-ls-1, which is more than a 2.4-fold lower than the rl for Gd-DOTA-dextran encapsulated into porous liposomes. Furthermore, the rl measured from Gd-DOTA- dextran encapsulated into liposomes is 2.6-fold higher than the rl of clinically used Gd-DOTA (3.9 mM-ls-1) . By conjugating cancertargeting ligands to their unobstructed outer surface, these CAs have the promise for use as targeted molecular imaging agents .

Kozlowska, D. et al. Gadolinium-loaded polychelating amphiphilic polymer as an enhanced MRI contrast agent for human multiple myeloma and non Hodgkin's lymphoma (human Burkitt's lymphoma) . RSC Adv. 4, 18007-18016 (2014) discloses the synthesis of liposomes loaded with Gd ions using different membrane-incorporated chelating lipids and functionalized with monoclonal anti-CD138 (syndecan-1) antibody for multiple myeloma and non-Hodgkin' s lymphoma diagnosis. In this case, the use of the polychelating amphiphilic polymer increases both the Gd content and the longitudinal relaxivity of the Gd-loaded liposomes as compared to Gd-DTPA-BSA equivalents . Park, J.-H. et al. Hyaluronic acid derivative-coated nanohybrid liposomes for cancer imaging and drug delivery. J. Control. Release Off. J. Control. Release Soc. 174, 98-108 (2014) discloses nanohybrid liposomes coated with amphiphilic hyaluronic acid- ceramide for targeted delivery of anticancer drug and in vivo cancer imaging. Dox, an anticancer drug, and Magnevist, a Gd-based CA for MRI, are loaded into this nanohybrid liposomal formulation. They find that in vitro release and in vivo clearance of Dox as well as cellular uptake from the nanohybrid liposome is enhanced than that from conventional liposome, thanks to the prolonged circulation of the nanohybrid liposome in the blood stream and to the HA-CD44 receptor interactions.

Huang, W.-C., Chen, Y . -C . , Hsu, Y.-H., Hsieh, W.-Y. & Chiu, H.-C. Development of a diagnostic polymersome system for potential imaging delivery. Colloids Surf. B Biointerfaces 128, 67-76 (2015) discloses a dual-imaging diagnostic polymersome system featured with highly hydrated multilamellar wall structure capable of simultaneously embedding a hydrophobic near-infrared fluorophore, Cy5.5 , and a paramagnetic probe, Gd cations. The polymersomes were obtained from the self-assembly of lipid-containing copolymer, poly(acrylic acid-co-distearin acrylate) , in aqueous solution. The Cy5.5 and Gd species were loaded into polymersomes via hydrophobic association (loading efficiency of Cy5.5 ca 74%) and electrostatic complexation (Gd ca 83%) , respectively. Owing to the highly hydrated structure of vesicular membrane, the superior contrast enhancement of Gd-loaded liposomes in MRI was obtained as a result of prolonged rotational correlation time of Gd cations and fast water exchange from Gd to bulk solution. The system exhibits a 15- fold higher longitudinal relaxivity value (ca 60 mM-ls-1) than that (4 mM-ls-1) of the commercial CA, Magnevist, in phosphate buffered saline. The in vivo characterization demonstrates that liposomes exhibit a signal-to-noise ratio in Tl-weighted MR image contrast similar to that of Magnevist, yet with a Gd dose 5-fold lower. Along with their non-toxicity at the dose used, these results demonstrate the great potential of the CGLPs as an advanced diagnostic nanodevice. An excellent contrast in NIR imaging at tumor site was attained following the intravenous inj ection of liposomes into Tramp-Cl tumor-bearing mice ( C57BL/ 6 ) .

Tian, B . et al . Mannose-coated gadolinium liposomes for improved magnetic resonance imaging in acute pancreatitis . Int . J . Nanomedicine 12 , 1127-1141 ( 2017 ) discloses the synthesis of Gd- DTPA-loaded mannosylated liposomes (M-Gd-NL ) and test their ability to target macrophages in Acute Pancreatitis (AP ) and discriminate between mild and severe AP . Lipid film-based method is used to synthesize DSPE-PEG2000-Man liposomes encapsulating DPPE-DTPA-Gd, with size around 100 nm . In vitro tests show efficient bind and readily release of Gd-DTPA into macrophages , resulting in enhanced MRI ability . Indeed, M-Gd-NL show a longitudinal relaxivity 1 . 8-1 . 9 higher than Gd-DTPA, as a consequence of the embedding of DPPE-DTPA-Gd into the bilayer of liposomes , which slowed down the tumbling motion of Gd complexes . As far as the safety profile , M-Gd-NL do not show any severe organ toxicity in rats , thus proving to be promising nanocarriers for clinical use and for the early detection of AP .

International patent Application, publication number W02017 / 093902 discloses a process for the preparation of nanoparticles of a cross-linked polysaccharide , inside said nanoparticles being geometrically confined a gadolinium-based or manganese-based contrast agent , said process comprising the steps of preparing a starting solution comprising the polysaccharide in a solvent and a gadolinium-based or manganese-based contrast agent , inj ecting said solution in a central channel of a microfluidic device comprising the central channel and side channels wherein an anti-solvent of said polysaccharide is inj ected, adding a crosslinking agent in the microfluidic device , precipitating nanoparticles consisting of cross-linked polysaccharide containing the contrast agent characterised in that the addition of the crosslinking agent is in the side channel containing the anti-solvent or, in the central channel containing the solution at a temperature comprised between 5 and 23 ° C and during precipitation the temperature is between 25 and 40 ° C and the ratio between the volumetric flow of the solution in the central channel and the volumetric flow of the i-solvent in the side channel ranges between 0.001 and 3.

International patent Application, publication number WO2018154470 discloses a process for the preparation of coacervate particles comprising the steps of preparing a water in oil emulsion of a biocompatible polyelectrolyte polymer and an aqueous solution of a biocompatible polyelectrolyte polymer having opposite charges of the polyelectrolyte of the in oil emulsion, adding two crosslinking agents, one to the emulsion and the other one to the aqueous solution and a contrast agent for medical imaging , mixing the aqueous solution with the emulsion at a temperature comprised between 19 and 37 °C and at a pH comprised between 3 and 7 to obtain the separation of the coacervate nanoparticles and optionally adding a further contrast agent, and optical tracer or a radiotracer for medical imaging to the coacervate particles .

International patent application, publication number WO2014187878 discloses liposomes comprising an aqueous gel core comprising nucleic acid fragments inside a lipid bilayer shell, wherein said particles can be obtained by using a microfluidic device, even if no details are given regarding the microfluidic process and/or device used. The size of microparticles may range from 1 to 200 pm and the lipid capsule can be a monolayer or a bilayer.

The lipid-polymer nanoparticles, that combine the biodegradable core of polymer with the lipid-layer or bilayer, have shown promising results in drug (Y.L. Zhang, P. Zhang, T. Zhu, Ovarian carcinoma biological nanotherapy: Comparison of the advantages and drawbacks of lipid, polymeric, and hybrid nanoparticles for cisplatin delivery, Biomedicine & Pharmacotherapy 109, 2019, 475- 483) and gene delivery applications (Z.M. Chen, F.Y. Liu, Y.K. Chen, J. Liu, X.Y. Wang, A.T. Chen, G. Deng, H.Y. Zhang, Z.Y. Hong, J.B. Zhou, Targeted Delivery of CRISPR/Cas 9-Mediated Cancer Gene Therapy via Liposome-Templated Hydrogel Nanoparticles, Advanced Functional Materials, 27 (46) , 2017) . Despite the potential of this next generation NPs (A. Mukherjee, A.K. Waters, P. Kalyan, A.S. Achrol, S. Kesari, V.M. Yenugonda, Lipid-polymer hybrid nanoparticles as a next-generation drug delivery platform: state of the art, emerging technologies, and perspectives, International Journal of Nanomedicine 14, 2019, 1937-1952) the employment of lipid-polymer NPs in microRNA delivery is still limited. Harsh temperatures, extreme pH, potentially toxic solvents and post-production steps required for micro-RNA processing pose a challenge for the preparation of NPs entrapping microRNA.

Conventional technique for liposome production such as thin film hydration technique and double emulsion methods, requiring sonication, extrusion trough the porous filter, centrifugation and gel column filtration to reduce the NPs size, polydispersity and to remove lipids aggregates or free nucleic acids, submit the microRNAs to potential degradation. Moreover, the microRNA higher affinity with water makes them quickly diffuse in water phase during the nanoprecipitation and emulsion- based preparation, results in a low encapsulation efficacy in lipid/polymer NPs. Furthermore, the multiple post processing steps, the need of large volume of costly materials, the low control over the final nanocarrier properties and the batch-to-batch variations strongly limit the NPs clinical translation (S.J. Shepherd, D. Issadore, M.J. Mitchell, Microfluidic formulation of nanoparticles for biomedical applications, Biomaterials, 274, 2021) . In contrast, microfluidics, manipulating continuously fluids at micrometer scale, have fine control over process parameters , so reducing the potential risk for microRNAs degradation and producing particles with tunable features, including size distribution, active agent loading and their release rate. Moreover, microfuidics limit the consume of raw materials and can be easy scale-up through device parallelization (S.J. Shepherd, C.C. Warzecha, S. Yadavali, R. El- Mayta, M.G. Alameh, L.L. Wang, D. Weissman, J.M. Wilson, D. Issadore, M.J. Mitchell, Scalable mRNA and siRNA Lipid Nanoparticle Production Using a Parallelized Microfluidic Device, Nano Letters 21(13) , 2021, 5671-5680; P.M. Valencia, O.C. Farokhzad, R. Karnik, R. Langer, Microfluidic technologies for accelerating the clinical translation of nanoparticles, Nature Nanotechnology, 7 (10) , 2012, 623-629. Staggered herringbone micromixers, inducing millisecond mixing of ethanol-lipid and nucleic acid -buffer streams, were used to produce LNP siRNA systems that result in smaller and more homogenous NPs than pipette mixing (N.M. Belliveau, J. Huft, P.J.C. Lin, S. Chen, A.K.K. Leung, T.J. Leaver, A.W. Wild, J.B. Lee, R.J. Taylor, Y.K. Tam, C.L. Hansen, P.R. Cullis, Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA, Molecular Therapy-Nucleic Acids, 1, 2012) . and high gene silencing in vivo (] D.L. Chen, K.T. Love, Y. Chen, A. A. Eltoukhy, C. Kastrup, G. Sahay, A. Jeon, Y.Z. Dong, K.A. Whitehead, D.G. Anderson, Rapid Discovery of Potent siRNA- Containing Lipid Nanoparticles Enabled by Controlled Microfluidic Formulation, Journal of the American Chemical Society, 134 (16) , 2012, 6948-6951) . Was also demonstrated that microfluidic potential to entrap active agent within lipid-NPs can be extended to macromolecules larger than siRNA, such as mRNA and DNA (A.K.K. Leung, Y.Y.C. Tam, S. Chen, I.M. Hafez, P.R. Cullis, Microfluidic Mixing: A General Method for Encapsulating Macromolecules in Lipid Nanoparticle Systems, Journal of Physical Chemistry, B 119(28) , 2015, 8698-8706; Y. Cao, Y.F. Tan, Y.S. Wong, M.W.J. Liew, S. Venkatraman, Recent Advances in Chitosan-Based Carriers for Gene Delivery, Marine Drugs, 17 (6) , 2019) .

Technical problem

In view of the findings of the prior art, the inventors of the present application designed a process for the preparation of hybrid nanostructures consisting of a lipid bilayer shell wrapping a hydrogel core wherein imaging agents and drugs are encapsulated.

The hybrid nanostructure of the present invention represents an improvement to conventional drug-encapsulating liposomes since it couples the advantages of the lipid layer for entrapping lipophilic or hydrophilic drugs and of the hydrogel structure for boosting the performances of the loaded imaging agents.

More in particular, the control of the rate of precipitation of the hydrogel core can change the water entrapment improving the water exchange rate so that the overall developed structure exhibits a relaxivity augmentation in comparison with free metalchelates and enhances the relaxometric properties of the encapsulated contrast agent from 1 to 10 times .

On the other hand, the lipid bilayer of the liposome prevents from leaking of the contrast agents , favours the biocompatibility of the nanostructure and its interactions with the surrounding bioenvironment .

Through the microfluidics is possible to finely control the coupling between the lipid bilayer fragments and the polymer core . Indeed, the microfluidics allows the control of two competitive mechanisms : the nanoprecipitation of the polymer in the mainstream and the formation of the lipid fragments that will provide the coating of the polymer structure .

The process of the present invention uses a microfluidic device and thanks to the purposive selection of all the working parameters , such as temperatures , flow rate and concentration of the materials , allows to obtain a final product with improved properties .

Considering the document WO2014187878 as the closest prior art , the present invention differs for the following characterising features : the particles have a bilayer lipidic shell with a cholesterol/ phosphatidylcholine molar ratio from 0 to 90% structure , obtained thanks to a microfluidic process .

The particles size is of the order of nanometers , a thousand times smaller than the micrometric particles of WO2014187878 . Such a difference in size , at these dimensional scales , implies the resolution of very different technical problems , which are frequently not comparable to each other .

Moreover, besides the nanometric size , thanks to the microfluidic platforms , the selection of the particle ' s diameter takes place during the production process , avoiding the use of additional steps to select particles with the desired size , such as centrifugation . Another, significant difference with WO2014187878 is that the nanoparticles are self-assembled .

The particles are obtained thanks to the specific design of the microfluidic process and the device thereof .

Unity of the Invention

The single general concept which is in common with all the aspects of the present application is the design of the microfluidic process and apparatus thereof , leading to obtain the specific particles with improved properties .

Object of the Invention

With reference to the attached claims , the above technical problem is solved by providing : a process for the preparation of self-assembled nanoparticles consisting of a lipid bilayer shell and a hydrogel core wherein active agents are entrapped, wherein the process is carried out in a microfluidic device comprising a central (middle ) channel and at least one side channel , the process comprising the following steps : a ) Preparation of a lipid stock solution made of at least two different lipids ; b ) Preparation of a solvent mixture by adding the lipid stock solution as obtained in step a ) to a solvent solution of at least one alcohol and water; c ) Preparing an aqueous solution of at least one hydrophilic polymer and the active agent and at least one solvent bearing at least one carboxylic group; d ) Inj ecting the solution as obtained in step c ) in the middle channel of the microfluidic device ; e) Injection the solution as obtained in step b) in a side channel of the microfluidic device; f) Collecting the final product as precipitate ad the confluence point of the middle and side channels;

Wherein in step a) the molar ratio between the two different lipids is comprised between 1:9 and 9:1; in step b) the solvent solution is at least one alcohol between 25% and 100% and water between 0% and 85%; in step c) aqueous solution contains an amount of at least one solvent bearing at least one carboxylic group lower than 30%; the flow rate in the middle channel is between 0.1 and 1000 pl/min .

A further object of the present invention are self-assembled nanoparticles consisting of a lipid bilayer shell and a hydrogel core characterised in having an average diameter comprised between 10 and 950 nm, polydispersity index comprised between 0,1 and 1, preferably obtained by the above process, the nanoparticles obtained by a process carried out in a microfluidic device comprising at least a central (middle) channel and at least one side channel comprising the following steps: a) Preparation of a lipid stock solution made of at least two different lipids; b) Preparation of a solvent mixture by adding the lipid stock solution as obtained in step a) to a solvent solution of at least one alcohol and water; c) Preparing a aqueous solution of at least one hydrophilic polymer and the active agent and at least one solvent bearing at least one carboxylic group; d) Injecting the solution as obtained in step c) in the middle channel of the microfluidic device; e ) Inj ection the solution as obtained in step b ) in a side channel of the microfluidic device ; f ) Collecting the final product as precipitate ad the confluence point of the middle and side channels ;

Wherein in step a ) the molar ratio between the two different lipids is comprised between 1 : 9 and 9 : 1 ; in step b ) the solvent solution is at least one alcohol between 25% and 100% and water between 0% and 85% ; in step c ) aqueous solution contains an amount of at least one solvent bearing at least one carboxylic group lower than 30% ; the flow rate in the middle channel is between 0 . 1 and 1000 pl/min .

Another obj ect of the present invention is the use of the above nanoparticles as carriers entrapping active ingredients .

Another obj ect of the present invention is the use of the above nanoparticles as contrast agents for imaging techniques .

Another obj ect of the present invention is the use of the above nanoparticles as drugs for theranostic applications .

Further features will be clear from the following detailed description with reference to the experimental data provided and the attached figure .

Brief description of figure

Figure 1 show in graph the results of relaxometry, distribution of magnetization longitudinal relaxing time T1 .

Detailed description of the invention

Definitions

Within the meaning of the present invention microfluidic process means a process carried out in a microfluidic devices provided with micro-channels wherein the flow of starting solutions at specific flow rates towards an outlet of the device allows obtaining precipitation of particles of nanometric size .

Within the meaning of the present invention hydrogel means a network of crosslinked hydrophilic polymer chains with water being the dispersion medium .

Within the meaning of the present invention active agent means imaging agents and drugs .

Within the meaning of the present invention "Self-assembly" is a process in which a disordered system of pre-existing components forms an organized structure or pattern as a consequence of specific, local interactions among the components themselves , without external direction .

Within the present invention the terms "central" and "middle" are synonyms .

Within the meaning of the present invention the term "Hydrodynamic Flow Focusing" ( HFF ) is an hydrodynamic flow that occurs when fluids with different velocities are inj ected side by side in a microfluidic platform.

Imaging agents can be fluorophores for optical imaging, metalchelates for Magnetic Resonance Imaging , radiotracers for Positron Emission Tomography .

Drugs can be lipophilic drugs and hydrophilic drugs .

The present invention concerns a process for the preparation of self-assembled nanoparticles consisting of a lipid bilayer shell and a hydrogel core acting as carriers wherein active agents are entrapped, wherein the process is carried out inn a microfluidic device comprising a central (middle ) channel and at least one side channel , the process comprising the following steps : a ) Preparation of a lipid stock solution made of at least two different lipids ; b) Preparation of a solvent mixture by adding the lipid stock solution as obtained in step a) to a solvent solution of at least one alcohol and water; c) Preparing an aqueous solution of at least one hydrophilic polymer and the active agent and at least one solvent bearing at least one carboxylic group; d) Injecting the solution as obtained in step c) in the middle channel of the microfluidic device; e) Injection the solution as obtained in step b) in a side channel of the microfluidic device; f) Collecting the final product as precipitate ad the confluence point of the middle and side channels;

A further object of the present invention are the self-assembled nanoparticles consisting of a lipid bilayer shell and a hydrogel core characterised in having an average diameter comprised between 20 and 950 nm, polydispersity index comprised between 0,1 and 1, the nanoparticles are obtained by a process carried out in a microfluidic device comprising a central (middle) channel and at least one side channel comprising the following steps: a) Preparation of a lipid stock solution made of at least two different lipids; b) Preparation of a solvent mixture by adding the lipid stock solution as obtained in step a) to a solvent solution of at least one alcohol and water; c) Preparing an aqueous solution of at least one hydrophilic polymer and the active agent and at least one solvent bearing at least one carboxylic group; d) Injecting the solution as obtained in step c) in the middle channel of the microfluidic device; e) Injection the solution as obtained in step b) in a side channel of the microfluidic device; f) Collecting the final product as precipitate ad the confluence point of the middle and side channels;

The nanoparticles are self-assembled .

Preferably the self-assembled nanoparticles consisting of a lipid bilayer shell and a hydrogel core characterised in having an average diameter comprised between 20 and 950 nm, polydispersity index comprised between 0,1 and 1, acts as carriers entrapping active agents.

Preferably, the self-assembled nanoparticles have a Zeta potential comprised between -42 and + 53 mV. More preferably, the self-assembled nanoparticles have a Zeta potential comprised between -22 and + 53 mV .

Preferably the self-assembled nanoparticles have mobility parameters comprised between -2 and -4 pmcm/Vs .

Preferably the self-assembled nanoparticles have conductivity comprised between 0 , 07 and 1 mS/cm .

Advantageous , Hydrodynamic Flow Focusing ( HFF ) is used to produce a complex Lipid-Polymer nanosystem named Lipid-polymer nanoparticles .

The HFF features govern the competition of two solvent extractions and therefore coordinate the relative kinetics of nuclei and growth of two techniques : nanoprecipitation and self-assembly . These mechanisms cannot be finely controlled by traditional processes ( such as batch systems , film hydration, etc . ) but the manipulation can be obtained by microfluidics .

Preferably, said microfluidic platform has a central (middle ) channel with diameter ranging from 100 M to 5 mm and length up to 20 cm .

Preferably, said microfluidic platform has a side channel with diameter ranging from 100pm to 2500 pm and length L up to 5 cm.

Preferably the alcohol is selected from the group consisting of : ethanol , methanol , acetone , isopropanol .

More preferably the alcohol is ethanol .

Preferably the solvent bearing at least one carboxylic group is selected from the group consisting of : acetic acid, adipic acid, formic acid, lactic acid, malic acid, propionic acid, succinic acid .

More preferably the solvent bearing at least one carboxylic group is acetic acid .

Preferably in step a ) the molar ratio between the two different lipids is 1 : 4 . Preferably in step a) lipids are phosphatidylcholine and cholesterol in a ratio 1:4.

Preferably in step b) the solvent solution is ethanol 65% and water 35%.

Preferably step b) in carried out at room temperature.

Preferably in step c) aqueous solution contains 1% of acetic acid.

Preferably in middle channel the flow rate is 3 pl/min.

Preferably in the side channels the flow rate is 42 pl/min.

Preferably lipids are phospholipids and sterols .

Preferably lipids are selected from the group consisting of Phosphatidylcholine (PC) , L-a phosphatidylcholine (Soy/Egg) (Soy- PC/Egg-PC) , hydrogenated soyphosphatidylcholine (HSPC) , Distearoylphosphatidylcholine (DSPC) ,

Dimyristoylphosphatidylcholine (DMPC) ,

Dimyristoylphosphatidylglycerol (DMPG) ,

Dipalmitoylphosphatidylcholine (DPPC) , PCs combined with polyethylene glycol (PEG) , PCs combined with fatty acid chains such as oleic, lauryl, palmitic and stearic acid, Phosphatidylglycerol (PG) , Cyclic complexes like Cyclodextrin, Phosphatidylserine (PS) , Sphingomyelin (SM) , Phosphatidic acid (PA) , Phosphatidylethanolamine (PE) , even more preferably phosphatidylcholine and cholesterol.

Preferably the hydrophilic polymer of the hydrogel is selected from the group consisting of polyelectrolytes, polysaccharides, glycosaminoglycans .

More preferably the hydrophilic polymer of the hydrogel is selected from the group consisting of hyaluronic acid, poly ( lactic-co-glycolic acid) , dextran, alginate, cellulose, chitosan, even more preferably is chitosan.

More preferably chitosan is in a concentration between

and 0.5 mg/ml . Even more preferably chitosan is in a concentration of 0.1 mg/ml.

Preferably acetic acid is in an amount of 1% of the aqueous solution .

In a preferred embodiment phosphatidylcholine and cholesterol in a ratio of cholesterol to phosphatidylcholine of 1:4, added to solution of 65% EtOH in 35% MilliQ water. Chitosan solution was adding step by step to 1 % acetic acid in water step by step added stirring all time at 25 °C. Flow rate in the side channels and flow rate in the middle channel 3 pL/min.

Another object of the present invention is the use of the above nanoparticles as carriers entrapping active ingredients .

Active ingredients are selected from the group consisting of : drugs, pharmaceutically active ingredients, imaging agents.

Preferably drugs are selected from the group consisting of chemotherapeutic drugs, DNA containing drugs, RNA containing drugs .

Another object of the present invention is the use of the above nanoparticles as contrast agents for imaging techniques .

Another object of the present invention is the use of the above nanoparticles as drugs for theranostic applications .

Preferably imaging agents are selected from the group consisting of optical tracers, dyes, radiotracers, contrast agents.

Preferably optical tracers and dyes are selected form the group consisting of: rhodamines-based dyes, bodipy-based dyes, indocyanines-based dyes, indocyanine green dyes, porphyrines-based dyes, phthalocyanines-based dyes.

Preferably Radiotracers are selected form the group consisting of: 99mTc based radiotracers, 18F based radiotracers, 18F-FDG radiotracers .

Preferably contrast agents are selected from the group consisting of: iodine-based contrast agents, barium-based contrast agents, iron-based contrast agents, gadolinium-based contrast agents, manganese-based contrast agent.

In a preferred embodiment the nanoparticle is made of a shell of phosphatidylcholine and cholesterol and a core made of chitosan entrapping Gadolinium- or Manganese-based contrast agent.

Examples

Example 1

Materials and methods

As lipids, was used phosphatidylcholine get from soybean (lyophilized powder; storage temperature -20°C; average molecular weight of approximately 776 g/mol) and cholesterol derived from sheep wool (Empirical Formula C27H46O; molecular weight 386.65 g/mol; storage temperature -20°C) from Sigma Aldrich. As polymer, was used chitosan (low molecular weight: 50,000-190,000 Da; soluble in dilute aqueous acid) Hyaluronic Acid, Poly Ethylene glycol (PEG) ; Polyethylenimine (PEI) ; poly ( lactic-co-glycolic acid) that we bought from Sigma Aldrich too. As solvents, were used acetic acid (absolute) , ethanol (puriss. p.a., absolute, b99.8%GC; MW: 46.07 g/mol) and filtered MilliQ water for all the experiments and purification steps (dialysis) .

Preparation of stock solution. Preparation of lipids stocks solutions (PC:Ch - 4:1) : PC: 80 mg in 10 ml (8 mg/ml) ; Ch: 10 mg in 10 ml (1 mg/ml) . At each trial ratio of cholesterol to phosphatidylcholine was 1:4, respectively.

To determine size distribution of NPs (hydrodynamic size and polydispersity index) , showed in table 4, dynamic light scattering (DLS) are performed. All samples are diluted (1:10) with deionized water to prevent the effects of multiple scattering. The measurement temperature is set at 25 °C.

Solubility tests

At first was prepared solution of lipids and ethanol, and then step by step adding 1% acetic acid in water (2.92ml water + 0.08 ml acetic acid) , solution was stirring all time (300x) at temp. 25 °C. Starting concentration of lipids in ethanol was 2.25 mg/ml after addition 3 ml of 1% acetic acid precipitation occur (1.28 mg/ml lipids concentration) . Solubility of lipids in ethanol and acetic acid was checked. Starting lipids concentration was 1, 6 mg/ml in ethanol. Then step by step acetic acid was added until 7.41% of acetic acid when precipitation occur. Precipitation disappears after decreasing acid percentage to 6% . In this way we checked solubility of lipids in presence of acetic acid (final lipids concentration 1.2 mg/ml) . With starting concentration of lipids 0.1125 mg/ml, 10% of acetic acid was obtained without precipitation, in the same solution solubility of chitosan was checked in concentration 0.25 mg/ml, 24h stirring, solution not transparent. Water was added (33.3%) to the solution step by step, after next 24h solution become transparent (lipids 0.00375 mg/ml; acetic acid 6.66%; ethanol 58.5 %; chitosan 0.166%; water 33.33%) . Next one test was solubility of chitosan in absolute acetic acid. Starting point was 5 mg/ml of chitosan in acetic acid; final concentration was 1 mg/ml, but solution was not transparent. For this reason, water was added, after 24h solution was transparent (final concentrations: acetic acid 77%; chitosan 0.77 mg/ml; water 23%) . The last one solubility test checked mixing of lipids solution (lipids: 0.072 mg/ml; ethanol 64.3%; water 35.7%) with chitosan solution (acetic acid 77 %; water 23%; 0.77 mg/ml) . Chitosan solution was adding step by step every 15 min to the lipids solution until following concentrations: lipids 0.05305mg/ml; chitosan 0.203 mg/ml; ethanol 45%; water 35%; acetic acid 20%.

Each trial was performed on microfluidic platform. Experimental conditions are reported in the following table 1.

Table 1

Characterizations of samples

Characterization was performed on transmission and Scanning electron microscope . Size of nanoparticles and Z-potential was analysed by dynamic light scattering ( DLS ) . Relaxation times are measured on a Philips MRI (magnetic field strength : 3 T ) . The acquisitions are performed at 37 ° C and, before each measurement , the sample is placed into the NMR probe for 15 min for thermal equilibration . Effect of EtOH concentration

Effect of two different EtOH volumetric fraction used in the solvent mixture to solubilize lipids ( 0 . 072 mg/ml ) was investigated . The optimal condition to make the self-assembly process effective can be observed in terms of liposomes production, morphology and size distribution . In particular, by solubilizing liposomes in a solvent mixture made of 65% EtOH and 35 % MilliQ water, lower size and polydispersity can be achieved after the processing through the microfluidic platform.

Effect of chitosan concentration

The effect of chitosan concentration on the liposomes formation and production was investigated . The best condition can be found at a chitosan concentration of 0 . 1 mg/ml . In fact , not only the size distribution is better, in terms of both size and polydispersity, than the trials with only acetic acid and the trial with 0 . 375 mg/ml , but also the structure of the liposomes seems to be able to encapsulate the polymer within the liposome core effect of acetic acid concentration .

The effect of increased acetic acid concentration on the liposome formation was investigated . In the case of 10% v/v acetic acid, the chitosan is no more entrapped into the liposome' s core .

Flow rates optimization

Was performed a comparison between two different flow rates used for the middle channel of the microfluidic platform. Chitosan- liposomes at 1 pL/min and the same liposomes obtained at 3 pL/min . The slightly higher flow rate in the middle channel represents not only an advantage in terms of higher throughput but also in terms of lower size and polydispersity .

Gd-DTPA encapsulation

Was performed a comparison between Gd-loaded chitosan-liposomes at two different flow rate conditions ( 1 pL/min and 3 pL/min ) . As already highlighted in the previous paragraph, the value of 3 pL/min is the best flow rate condition . Smaller and less polydisperse chitosan-liposomes encapsulating Gd-DTPA can be obtained with a middle channel flow rate set at 3 pL/min .

Moreover, in terms of longitudinal relaxation time (Tl ) , the best process condition is obtained at 3 pL/min flow rate in the middle channel . As showed in Table 2 and 3 , the Tl of Gd-loaded chitosan- liposomes , is far lower than that of the unloaded chitosan- liposomes . Furthermore , after a dialysis to remove the excess of Gd-DTPA, the signal for the Gd-loaded chitosan-liposomes is still significant , meaning that the Gd-DTPA is encapsulated into the liposomes core .

Table 2

Table 3

In the following table 4 data concerning average nanoparticles ' diameter and Polydispersity index ( PDI ) are reported . Among the best achieved conditions , the one highlighted in bold in table 4 represent the size distribution of unloaded and Gd-loaded chitosan-liposomes at two different flow rate conditions ( 1 pL/min and 3 pL/min ) . As already highlighted, the value of 3 pL/min is the best flow rate condition even in the case of Gd-loaded liposomes . Smaller and less polydisperse chitosan-liposomes encapsulating Gd-DTPA can be obtained with a middle channel flow rate set at 3 pL/min . Table 4

Example 2

Materials and Methods

The nanoparticles of the present invention underwent to further experimentation .

L-a-Phosphatidyl choline from soybean X99 % (SPC; lyophilized powder; storage temperature -20°C; approximately Mw= 776 g/mol) and Cholesterol b99 % (Choi; powder; storage temperature -20°C; Empirical Formula C27H46O; Mw = 386.65 g/mol) have been purchased from Sigma-Aldrich (St. Louis, MO, USA) . Chitosan (CH; powder; Low Mw = 50,000-190,000 Da; soluble in dilute aqueous acid) ;

Diethylenetriaminepentaacetic acid gadolinium (III) dihydrogen salt hydrate (Gd-DTPA; Mw = 547.57 g/mol) , Atto 633 (Xex/em= 633/657 nm, Mw = 652 g/mol) and Atto 488 (Xex/em= 504/521 nm, Mw = 804 g/mol) have been purchased from Sigma Aldrich ( St. Louis, MO, USA) . Irinotecan HC1 Trihydrate (IRI/Antimir 21, Mw = 667.18 mg/ml, Xabs= 368 nm) was purchased by Selleckchem Chemicals (Huston, USA, ) . As solvents, we have used Acetic acid glacial (AcOH, X99.8%; Empirical formula CH₃COOH; Mw =60.052 g/mol, ROMIL pure chemistry, Cambridge, UK) , Ethanol (EtOH, puriss. p.a. , absolute, X99.8%GC; Empirical formula C2H5OH; MW: 46.07 g/mol; Carlo Erba Reagents, Italy) and filtered MilliQ water (Milli-Q Plus, Q-POD®, Merck KGaA, Darmstadt, Germany) for all the experiments. The phosphate buffer saline (PBS, tablet) for dialysis, cell-culture and in vitro studies was purchased by Sigma Aldrich (St. Louis, MO, USA) . CellMask Orange Plasma membrane Stain (Xex/em=554 /567 nm) was purchased from Thermofisher

Scientific (Altrincham, UK)

Microfluidic set-up for flow focusing approach

A quartz microfluidic device (22.5mm long x 15mm wide x 4mm thick) with 5 parallel inputs and one output purchased from Dolomite Centre Ltd (Royston, UK) , is used to perform all the experiments. The device consists of 5 parallel inlets converging and intersecting the corresponding end of the central channel at an angle of 45°at the junction, followed by a straight output channel. All channels have the same approximately circular cross- section of 160 x 150 pm. Only three of five inlets of the device are used for Lipid-polymer nanoparticles production. The chip is compatible with the H interface 7-way for tubing connections. The device is connected to 2.5-5/10 mL glass syringes with two PTFE tubing segments (1/16' x 0.25 nm- 0.8 x 0.25 mm) controlled by a low-pressure syringe pump. Two-way in line ETFE valves, connecting syringes with the microfluidic device, make the automatic fill-in of the syringes feasible, thus allowing a continuous dispensing of reagents. A PTFE outlet tube (0.8 x 0.25 mm) , that starts from output of the device, is employed to collect fluid in a glass vial containing water.

One step HFF for Lipid-polymer nanoparticles production

A microfluidic process is used to produce a complex nanostructure named Lipid-polymer nanoparticles. The first step consists in the preparation of EtOH-Water solution (65% v/v- 35 % v/v) containing 0.0072 % w/v of lipids (mass ratio 8:1- SPC:Chol) . It is kept under continuous stirring overnight and then injected through the side channels. The Water phase is made of an aqueous solution 0.01% w/v of CH and 1% v/v of AcOH . It is kept under continuous stirring for at least 1 h and then injected through the middle channel. In the preparation of Gd-DTPA loaded Lipid-polymer nanoparticles, the contrast agent at a concentration of 0.4 % / r, is added to the acid solution containing chitosan (0.01 %w/v) . Atto 633, Atto 488 and Irinotecan co-encapsulation is achieved by dissolving the active agents in the acetic acid solution containing AcOH-CH-Gd-DTA (1 % v/v- 0.01 % w/v- 0.4 % w/v) .The Atto 633, Atto 488 and Irinotecan concentration were 24 ug/ml, 32.2 ug/ml and 145 ug/ml, respectively. Different flow rates have been tested, and the optimal Flow Rate Ratio FR2 (0.073) , defined as the ratio of Volume Flow Rate of middle channel (3 pl/min) and Volume Flow Rate of side channel (41 pl/min) , is determined for all formulations. The microfluidic process is carried out 40 min or its multiples and the nanoparticles are collected in a vial glass containing 3.5 mL of water or its multiples. The suspension was stirred for 40 min at room temperature. Each experiment has been repeated at least ten times . Lipid-polymer nanoparticles Physicochemical characterization

Dynamic light scattering (DLS) is used to determine nanoparticle size. The wavelength of the laser is 633 nm and the scattering angle used is 173° . The volume of the sample suitable for DLS analysis is 1 ml in a polystyrene cuvette. In DLS analysis, the z- Average value and the polydispersity index of the average of three measurement is collected. The DLS analysis were performed at 25 °C or 37 °C according to the aim of analysis. Zeta potential measurements are also performed at a temperature of 25 °C

In vitro MRI .

The relaxometric properties of blank nanoparticles (Lipid-polymer nanoparticles) and nanoparticles containing Gd-DTPA (Gd-DTPA Lipid-polymer nanoparticles, Atto 633 loaded Lipid-polymer nanoparticles (Atto633 Lipid-polymer nanoparticles) , Atto 633 coloaded Gd-DTPA Lipid-polymer nanoparticles (Atto633-Gd-DTPA Lipid- polymer nanoparticles) , Irinotecan-Gd-DTPA co-loaded Lipid- polymer nanoparticles (IRI-Gd-DTPA Lipid-polymer nanoparticles) ) are tested by in vitro MRI. The data are compared with Gd-DTPA calibration curves dispersed in water and in water-ethanol (70% w/w- 30% w/w) ranging from 0 to 100 uM and 0 to 120 uM, respectively. 300 pl of NPs and diluted NPs suspension in water (1:2) are dropped to NMR tube and the changes in relaxation time (Tl) are evaluated at 1.5 Tesla at 37 °C.

The Hydrodynamic Flow Focusing (cHFF) is used to produce a complex Lipid-Polymer nanosystem. The control of two phenomena is obtained simultaneously: the polymer nanoprecipitation, control-ling the time scales of solvent exchange and the self-assembly of bilayer fragments that decorate the NPs surface. The unique approach for the synthesis of Lipid-polymer nanoparticles relies on the injection of two lateral lipids streams, dissolved in variable ethanol-water (EtOH-Water) ratio, that squeeze the chitosan (CH) , dissolved in acetic acid (AcOH) solution, injected in the middle channel .

The HFF features govern the competition of two solvent extractions and therefore coordinate the relative kinetics of nuclei and growth of two techniques : nanoprecipitation and self-assembly . The steps of HEE involve a rapid nucleation rate of chitosan, selfassembly of lipids in bilayer fragments and, then, finally, the coupling of chitosan with the bent bilayer fragments . Consequently, the rapid chitosan precipitation mediates the bilayer fragments enclosure . Indeed, the designed HEE leverages rapid acetic acid (AcOH ) extraction that promotes fast nucleation, leading to almost monodisperse chitosan nanoprecipitate . Simultaneously, once the lateral solution comes in contact with the middle flow, the lipids are no longer solubilized and begin assembly in bilayer fragments due to the organic solvent extraction . Then, the coupling occurs and the already formed bilayer fragments ( slightly negatively charged) diffuse to the polymer nuclei nanoparticles (positively charged ) , covering their surface and so inhibiting the further growth of chitosan nuclei , finally stabilizing the Lipid-polymer nanoparticles complex . The process parameters were investigated, in terms of fine-tuning of the flow rates , solvent-non solvent ratio , solute concentration and ER² , that govern the coupling time of thermodynamic phenomena : nucleation of chitosan particles , self-assembly of lipid fragments and coupling of these intermediate structures . In this work the FR2 is defined as follows :

Pj^₂ Volume Flow Rate of AcOH- Water solution (Middle phase) Volume Flow Rate of EtOH- Water solution(Side phase)

A preliminary study was performed evaluating the effect of solvent-non solvent ratio ( EtOH/Water range : 80%/20 % v/v -65% /35% v/v) and the concentration of the reagents ( range of chitosan concentration : 0 . 01 % w/v-0 . 0375% w/v) on the morphology of Lipidpolymer nanoparticles and their physiochemical properties . The stability of the microfluidic process and the absence of massive precipitation, combined with the evaluation of the morphologies obtained by TEM images , were used to identify the best parameters as standard conditions for further optimization . The best condition was set to 0.0072 % w/v of Lipids (mass ratio 8:1- SPC:Chol) dissolved in EtOH-Water (65 % v/v -35 % v/v) , while the 0.01 % w/v of chitosan was dissolved in acid solution (1% v/v) .

Next was investigated the role of solvent displacement and residence time distribution on the morphology of nanoparticles by manipulating the FR². Variation of FR² from 0.024 to 0.17 was obtained by ranging middle channel flow rates from 1 pl/min to 7 pl/min (with constant stepwise of 2 ul/min) , while side channel ones were kept constant at 41 pl/min. By changing the middle flow rate, the consequent increase of flowrate ratio reduces the chitosan entrapment within the Lipid-polymer nanoparticles complex as confirmed by the positive zeta potential (44.2 mV) for the higher flowrate ratio (0.17) . We observe the formation of chitosan precipitates as uncontrolled morphologies and polymer-coated structures .

Further studies on FR², were conducted and results were compared in terms of z-Average, Particle Size Distribution (PSD) , Polydispersity Index (PDI) and Standard Deviation (St. Dev) value and Zeta potential.

By decreasing the FR² from 0.073 to 0.024, we observe an increase in the nanoparticle z-average size, from 77.47 nm to 261.1 nm, and increasing in nanoparticles polydispersity, as shown by PSD. The decreasing of the FR² leads to a reduction of the T_mlx, of the components, enhancing the chitosan nuclei formation but slowing down the phospholipid's bilayer assembly. We hypothesized that the polarity of the environment at the microfluidic junction is enough to induce a bilayer fragments formation and their bending, but not their closure. Downstream of the focusing region, the solvent extraction cannot induce a rapid closure of the bilayer. Still, it will occur later along the microfluidic chip length or collection volume. Indeed, a certain/adequate time is required for lipid fragments to aggregate. This slow gradient results in the growth phase of intermediate complexes.

By further reducing the FR², the production moves towards a region where every variation would not produce any effect different from massive aggregate precipitation of chitosan and uncontrolled growth of bilayer fragments . This effect is confirmed by TEM images , for the lowest FR² value a large liposome structure surrounded by some chitosan precipitate , and a a monodisperse population of Lipid-polymer nanoparticles for FR² value of 0 . 073 . The TEM image shows a nanostructured system, where is recognizable a dark core due to the polymeric entrapment within the liposome vesicle .

Was investigated the influence of the collection volume on liposome morphology at fixed FR2 of 0 . 073 . DLS measurement and TEM images show an enlargement of Lipid-polymer nanoparticles size as the collection volume increases up to 8 ml . The rapid change in pH around the liposome , as they are produced, could lead to a diffusion of water inside the liposome to balance the pH difference created between the newly formed chitosan core and neutral pH of the collection volume . Moreover , at the lower collection volume of 3 . 5 ml , the high residual ethanol amount could decrease the water permeability by replacing the water in the hydration shells of the head groups and accumulating itself in transient defects in the hydrophobic part of the bilayer . The assumption perfectly matches the irregular and swelled shape of the Lipid-polymer nanoparticles observed in the TEM images .

As a result of optimization studies for Lipid-polymer nanoparticles synthesis , the value of FR² of 0 . 073 , obtained at middle flow rate of 3 ul/min and side flow rate of 41 ul/min, the collection volume of 3 . 5 ml , chitosan concentration of 0 . 01 % w/v and Lipids concentration of 0 . 0072 % w/v proved to be the optimal conditions .

The mean and the mode of nanoparticle size are 94 . 4nm and 76 . 9nm, respectively, with 90% of the nanoparticles being <137 . 116 . 0 nm. The real-time visualization of Lipid-polymer nanoparticles as individual particles confirmed their stability in PBS . The nanoparticle concentration is around 1 . 24 x elO particles/ml . Results clearly show that Lipid-polymer nanoparticles are monodisperse NPs and stable upon aggregation phenomena . Stress testing of unloaded liposomes are conducted to evaluate possible degradation due to hydrolysis of saturated and unsaturated lipids . The Lipid-polymer nanoparticles size distribution was evaluated by DLS over time (up to 13 h) at 37 ° C and no significant increase in their average size and St . Dev was observed, the electrostatic interaction between the chitosan entrapment and lipid bilayer reduces the phosphate group ' s motional freedom, increasing the stability .

The designed microfluidic process and the Lipid-polymer nanoparticles core-shell structure make this set-up suitable for active compound loading . Indeed, the payload-agents are dissolved in chitosan solution and then inj ected in the middle channel , at optimal conditions , so forcing their loading in the core of Lipidpolymer nanoparticles complex . Firstly, the diagnostic properties of Gd-DTPA loaded Lipid-polymer nanoparticles (Gd-DTPA Lipidpolymer nanoparticles ) were studied by adding the contrast agent to the central polymer solution (mass ratio 1 : 40- CH : Gd-DTPA) of the microfluidic platform. Higher precipitation along the focused stream without instabilities at flow focusing interface was observed .

A slight increase in size of Lipid-polymer nanoparticles with the loading of Gd-DTPA is outlined (Table 1 ) . The TEM image Figure 4 a shows a less stained core of nanoparticles resulting from Contrast Agent ( GA) loading within chitosan matrix . The in vitro longitudinal relaxation time T1 of Gd-DTPA Lipid-polymer nanoparticles is evaluated at 37 ° C and 1 . 5 T . The amount of Gd- DTPA entrapped in the NPs was quantified from a calibration curve by Philips MRI 1 . 5 tesla ( Figure S8 ) . The Gd-DTPA Lipid-polymer nanoparticles show an encapsulation efficacy of Gd-DTPA of 78 % ( Figure S8 A and Table 1 ) . Lipid-polymer nanoparticles show a slightly negative surface charge of - 17 . 4 mV that may be attributed to the replacement of a phospholipid by cholesterol (mass ratio 8 : 1- SPC : Chol ) [ 49 ] ; while Gd-DTPA Lipid-polymer nanoparticles display a zeta potential of - 11 mV linked to the encapsulation of Gd-DTPA ( Table 1 ) . Was examined the optical and theranostic properties of Lipidpolymer nanoparticles by simultaneous encapsulation of Gd-DTPA and active agents (Atto 633/ Irinotecan or antimir21 ) , alternatively . In the co-loading of Atto 633 , a decrease in the Gd-DTPA EE % from 78% to around 67% is observed .

The following Table 5 shows co-encapsulation of Irinotecan ( average size , standard deviation, zeta potential and coencapsulated efficacy for different Lipid-polymer nanoparticles formulations .

Table 5

The amount of co-loaded Atto 633 and Irinotecan/antimir21 has been quantified through Photometry starting from a calibration curve. The co-EE% of Irinotecan is 64%, while the co-EE % of Atto 633 is 55 %. No effect of co-loading of Gd-DTPA is reported on the Atto 633 loading efficacy (55 %) , with respect to Atto 633 alone (EE-57

%) . DLS measurements of Gd-DTPA Lipid-polymer nanoparticles and IRI-Gd-DTPA Lipid-polymer nanoparticles show sizes of 95.3 ± 26.35 nm, 112.8 ± 30 nm, respectively. No impact of the entrapment of active agents on surface charge of Lipid-polymer nanoparticles is reported, unless for the loading of Atto 633, a cationic dye, that reduces the Lipid-polymer nanoparticles surface charge (-3.7 mV) .

Claims

1. Process for the preparation of self-assembled nanoparticles consisting of a lipid bilayer shell and a hydrogel core wherein active agents are entrapped wherein the process is carried out in a microfluidic device comprising a central (middle) channel and at least one side channel, the process comprising the following steps: a) Preparation of a lipid stock solution made of at least two different lipids; b) Preparation of a solvent mixture by adding the lipid stock solution as obtained in step a) to a solvent solution of at least one alcohol and water; c) Preparing a aqueous solution of at least one hydrophilic polymer and the active agent and at least one solvent bearing at least one carboxylic group ; d) Injecting the solution as obtained in step c) in the middle channel of the microfluidic device; e) Injection the solution as obtained in step b) in a side channel of the microfluidic device f) Collecting the final product as precipitate ad the confluence point of the middle and side channels;

Wherein in step a) the molar ratio between the two different lipids is comprised between 1:9 and 9:1; in step b) the solvent solution is at least one alcohol between 25% and 100% and water between 0% and 85%; in step c) aqueous solution contains an amount of at least one solvent bearing at least one carboxylic group lower than 30%; the flow rate in the middle channel is between 0.1 and 1000 pl/min . Process according to claim 1 wherein in step a) the ratio between the two different lipids is 1:4.

3. Process according to claim 1 wherein in step b) the solvent solution is ethanol 65% and water 35%.

4. Process according to claim 1 wherein in step c) aqueous solution contains 1% of acetic acid.

5. Process according to claim 1 wherein in middle channel the flow rate is 3 pl/min.

6. Process according to claim 1 wherein lipids are phospholipids and sterols.

7. Process according to claim 1 wherein hydrophilic polymer of the hydrogel is selected from the group consisting of polyelectrolytes, polysaccharides, glycosaminoglycans.

8. Self-assembled nanoparticles consisting of a lipid bilayer shell and a hydrogel core characterised in having an average diameter comprised between 20 and 950 nm, polydispersity index comprised between 0,1 and 1.

9. The self-assembled nanoparticles according to claim 8 obtained by a process carried out in a microfluidic device comprising a central (middle) channel and at least one side channel comprising the following steps: a) Preparation of a lipid stock solution made of at least two different lipids; b) Preparation of a solvent mixture by adding the lipid stock solution as obtained in step a) to a solvent solution of at least one alcohol and water; c) Preparing a aqueous solution of at least one hydrophilic polymer and the active agent and at least one solvent bearing at least one carboxylic group ; d) Injecting the solution as obtained in step c) in the middle channel of the microfluidic device; e) Injection the solution as obtained in step b) in a side channel of the microfluidic device f) Collecting the final product as precipitate ad the confluence point of the middle and side channels; Wherein in step a) the molar ratio between the two different lipids is comprised between 1:9 and 9:1; in step b) the solvent solution is at least one alcohol between 25% and 100% and water between 0% and 85%; in step c) aqueous solution contains an amount of at least one solvent bearing at least one carboxylic group lower than 30%; the flow rate in the middle channel is between 0.1 and 1000 pl/min .

10. Use of nanoparticles according to claim 8 as carriers for entrapping active ingredients .