COMPLEXES FOR THE DELIVERY OF PROTEINACEOUS AGENTS

Abstract

The invention provides a complex comprising at least one proteinaceous agent, an ionic polymer comprising a repetitive unit of formula (I):

##STR00001##

wherein R.sup.1 represents a hydrogen atom or a straight or branched chain alkyl group, preferably a straight or branched chain alkyl group comprising from 1 to 6 carbon atoms, for example a methyl group; R.sup.2 represents a straight or branched chain alkyl group which is substituted by a group which may have a positive charge at a physiological pH; and optionally a surfactant; a complex for use in a method of medical treatment; a pharmaceutical composition

Claims

1. A complex comprising at least one proteinaceous agent, an ionic polymer comprising a repetitive unit of formula (I): ##STR00014## wherein R.sup.1 represents a hydrogen atom or a straight or branched chain alkyl group, preferably a straight or branched chain alkyl group comprising from 1 to 6 carbon atoms, preferably a methyl group; R.sup.2 represents a straight or branched chain alkyl group which is substituted by a group which has a positive charge at a physiological pH; and optionally a surfactant; with the proviso that when the at least one proteinaceous agent is heparin and when the complex does not include a surfactant, the ionic polymer is not a poly (dimethyl amino) ethyl methacrylate having a relative number-average molecular weight of 8,000 Da or 15,000 Da.

2. A complex as defined in claim 1 which comprises a surfactant.

3. A complex as defined in claim 1 wherein the ionic polymer comprises a single repetitive unit of formula (I), or the repetitive unit of formula (I) is combined with other monomers or other polymer or oligomeric sequences.

4. A complex as defined in claim 1 wherein the ionic polymer comprises repetitive units of formula (I) wherein at least some repetitive units of formula (I) are charged.

5. A complex as defined in claim 1 wherein the ionic polymer is an ionic polymer of formula: ##STR00015## wherein X.sub.1 and X.sub.2 each respectively represent the alpha and omega end groups of the polymer; R.sup.1 and R.sup.2 are each as defined in claim 1; and n represents the number of repetitive units of the ionic polymer.

6. A complex as defined in claim 1 wherein the ionic polymer has a mean pKa of from 6 to 8.

7. A complex as defined in claim 1 wherein the repetitive unit of formula (I) comprises N,N dimethyl amino ethyl methacrylate.

8. A complex as defined in claim 1 wherein the at least one proteinaceous agent comprises one or more proteinaceous agents.

9. A complex as defined in claim 1 wherein the at least one proteinaceous agent comprises an antigen.

10. A complex as defined in claim 1 which is encapsulated by an alginate coating.

11. A complex as defined in claim 1 wherein the molar ratio of (dimethyl amino) ethyl methacrylate to a different repetitive unit of formula (I) is greater than 50%.

12. A method of medical treatment, the method comprising administering to a human or animal in need of such treatment an effective amount of a complex comprising at least one medically or veterinary active proteinaceous agent, an ionic polymer comprising a repetitive unit of formula (I): ##STR00016## wherein R.sup.1 represents a hydrogen atom or a straight or branched chain alkyl group, preferably a straight or branched chain alkyl group comprising from 1 to 6 carbon atoms, preferably a methyl group; R.sup.2 represents a straight or branched chain alkyl group which is substituted by a group which has a positive charge at a physiological pH; or a copolymer thereof; and optionally a surfactant.

13. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a complex as defined in claim 1.

14. A method of medical or veterinary treatment, the method comprising administering a composition comprising a pharmaceutically acceptable carrier and a complex as defined in claim 12.

15. A method of medical treatment which method includes a step of administering to a human or animal in need of such treatment an effective amount of a complex which comprises at least one medically active proteinaceous agent, an ionic polymer comprising a repetitive unit of formula (I) as defined in claim 12 or a copolymer thereof; and a surfactant.

Description

[0085] The invention will now be illustrated with reference to the following Figures of the accompanying drawings which are not intended to limit the scope of the claimed invention:

[0086] FIG. 1 illustrates the results of Example 1 and shows graphs which illustrate the evolution of the mean light scattering Intensity (Id cpm) and mean diameter of the PAEC;

[0087] FIG. 2 illustrates the results of Example 1 and shows a comparison of an electrophoretic profile of free ovalbumin versus ovalbumin associated with ionic polymers, either OB006 (PDMAEMA with a Mw of 10 kDa) and DA002 (PDMAEMA with a Mw of 90 kDa);

[0088] FIG. 3 illustrates the results of Example 2 and shows graphs which illustrate the evolution of the mean light scattering Intensity (Id cpm) and mean diameter of the PAEC loaded with human insulin;

[0089] FIG. 4 illustrates the results of Example 2 and shows CD spectra analysis of free insulin or associated with an ionic polymer;

[0090] FIG. 5 illustrates the results of Example 2 and shows TEM images (20,000× magnification where the horizontal line on each image represents a distance of 1 μm at that magnification) of PAEC loaded with human insulin with and without PEG;

[0091] FIG. 6 illustrates the results of Example 4 and shows graphs illustrating the kinetics of formation of quaternary PAEC made from CpG, P24, SDS and an ionic polymer (DA002) controlling the mean light scattering intensity (Id) and the mean size of the PAEC suspension by dynamic light scattering (DLS);

[0092] FIG. 7 illustrates the results of Example 4 and shows a graph illustrating the stability of quaternary PAEC made from CpG, P24, SDS and an ionic polymer (DA002) made with P24 loading ranging from 50 to 250 μg/m L;

[0093] FIG. 8 illustrates the results of Example 4 and shows a comparison of the mean diameter measured by DLS (shown on y-axis by units of nanometres) of 6 different batches of PAEC loaded with respective concentrations of P24/CpG/SDS/ionic polymer DA002 were 200/80/200/350 μg/mL. The batch number 5 has also been submitted to 3 freeze-thawing cycles;

[0094] FIG. 9 illustrates the results of Example 4 and shows a comparison of the electrophoretic profile of free P24 with P24 associated with cationic polymers, either OB006 or DA002;

[0095] FIG. 10 illustrates the results of Example 4 and shows a comparison of the electrophoretic mobility of PAEC loaded with respective concentrations (μg/mL) of P24/CpG/SDS/ionic polymer DA002 in comparison to latex (track 13);

[0096] FIGS. 11A and 11B show TEM micrography images (5.000× magnification where the horizontal line on each image represents a distance of 500 nm at that magnification) which illustrate the results of Example 4;

[0097] FIG. 12 shows a bar chart which illustrates the results of Example 5 by mean light scattering intensity (Id) measured by DLS of PAECs;

[0098] FIG. 13 shows a bar chart which illustrates the results of Example 4 which are an analysis of the medium term stability of the PAEC loaded with CpG and P24 in a PBS medium at temperatures of 4, 25 or 37° C. The evolution with time of the mean light scattering Intensity has been analyzed up to 90 days;

[0099] FIG. 14 shows TEM micrography images (5.000×) which illustrate the results of Example 6 which are of alginate microbeads loaded with binary PAEC (a) and Alginate microbeads loaded with ternary PAEC stabilized with SDS (b);

[0100] FIG. 15 shows a bar chart which illustrates the results of Example 7 which are the mean size (measured by mean diameter (nm) on the y-axis) of three batches of PAEC;

[0101] FIGS. 16A and 16B each show a bar chart which illustrate the results of Example 8 which are the systemic immunological responses observed on mice after intranasal administration (IN) of quaternary PAECs. The systemic immunological response has been analysed monitoring in the blood the CD8+ T-cells producing IFNg, TNFa, and IL2 (FIG. 15A) and CD4+ T-cells producing IFNg, TNFa, and IL2 (FIG. 15B), Ag stands for antigen;

[0102] FIG. 17 shows a bar chart which illustrates the results of Example 8 which are the systemic immunological responses observed on mice after intranasal administration (IN) of quaternary PAECs, by monitoring the antibody P24 concentration in the bloodstream;

[0103] FIG. 18 shows a bar chart which illustrates the results of Example 8 which are the systemic immunological responses observed on mice after intramuscular administration (IM) of ternary or quaternary PAECs. The systemic immunological response has been analysed by monitoring in the blood the CD8+ T-cells producing IFNg, TNFa, and IL2 (FIG. 17a) and CD4+ T-cells producing IFNg, TNFa, and IL2 (FIG. 17b), Ag stands for antigen; and

[0104] FIG. 19 shows a bar chart which illustrates the results of Example 8 which are the systemic immunological responses observed on mice after intramuscular administration (IM) of quaternary or ternary PAECs analysed by monitoring the antibody P24 concentration in the bloodstream.

[0105] The invention will now be illustrated in the following Examples which are not intended to limit the scope of the claimed invention.

EXAMPLES

[0106] Our formulation was tested with several proteinaceous drugs, namely human insulin, ovalbumin or a combination of an antigenic protein and an immunoadjuvant such as an oligonucleotide. The formation and stability of these PAEC was analysed with the following methods: [0107] i. Dynamic Light Scattering (DLS) giving the mean light scattering Intensity (Id) and autocorrelation function evolution, indicates the formation of the complex and its size as well as stability of PAEC (ionic strength, time, mechanical solicitation) [0108] ii. Electrophoretic Light Scattering (Coulter Delsa) giving electrophoretic mobility [0109] iii. Electrophoresis conducted in non-denaturating conditions to evaluate drug loading after separation of PAEC from the free peptide/protein [0110] iv. HPLC to determine free surfactant concentration [0111] v. TEM microscopy to analyse the morphology of the PAEC [0112] vi. In vitro release kinetics of the proteinaceous drugs [0113] vii. Circular dichroism to control the conformation and hence stability of the proteinaceous drug

Example 1. Preparation of Binary Polyelectrolyte Complexes with Ovalbumin

[0114] PAEC is prepared by physical mixing of aqueous solutions at room temperature. The aqueous solutions are previously filtrated on sterile 0.2 μm filters and the whole formulation procedure is realized within a laminar flow. The proteinaceous drug is ovalbumin, a protein of chickens which consists of 385 amino acids with a relative molecular mass of 42.7 kDa and with a serpin-like structure in a native status and an isolectric point of 4.5 (P. E. Stein, A. G. W. Leslie, J. T. Finch; R. W. Carrell, Crystal structure of uncleaved ovalbumin at 1.95 Å resolution”. Journal of Molecular Biology 1991, 221 (3): 941-959). The ionic polymer is poly(2-dimethylamino)ethyl methacrylate (PDMAEMA), with a molecular weight from 7 kDa to 91 kDa) dissolved in a sodium phosphate buffer medium equilibrated at a pH of about 7.4. Its concentration is ranging between 0.1 to at least 10 mg/mL, depending on the final drug loading, typically 10 to 50 wt %. Ovalbumin is dissolved carefully in the same buffer solution.

[0115] The mixing of the ionic polymer and ovalbumin solutions is performed in a polypropylene tube. The protein solution is typically first added in the appropriate vessel and a given volume of ionic polymer solution is quickly added in one time with a suitable injection device, directly within the solution and not against the wall of the vessel. The volume ratio of protein solution to the ionic polymer solution ranges between 1:1 to at least 100:1 and is adjusted in order to afford a rapid and homogeneous distribution of the ionic polymer within the protein solution. Ionic polymer volume is for example 1/10 of the total volume of PAEC solution.

[0116] PAEC formation between ovalbumin and the ionic polymer has been verified by DLS, monitoring both the increase in mean light scattering intensity and the appearance of an autocorrelation curve.

[0117] The size distribution in intensity of the PAEC's have been calculated after deconvolution of the autocorrelation curves and disclosed a mean radius in Intensity of PAEC in the nanosize range.

TABLE-US-00001 TABLE 1A Mw Radius at 50% (nm) 7000 236 +/− 22 13000 248 +/− 13 26400 453 +/− 5  49100 5171 +/− 225 91400 2852 +/− 120

TABLE-US-00002 TABLE 1B Ionisation % Radius at 50% (nm) 30  248 +/− 13 47 353 +/− 7 60 210 +/− 8 80 222 +/− 6

[0118] Table 1A shows the evolution of the mean radius (percentile 50) of PAEC5 loaded with ovalbumin as a function of the Mw of ionic polymer keeping constant the ionisation percentage to 30%. Table 1B shows the percentage of ionic groups in relation to the total number of repetitive units in the ionic polymer of PAEC5 loaded with ovalbumin where the ionic polymer has a Mw of 13 kDa.

[0119] Different molecular weights (Mw) and charge density of ionic polymer were tested keeping all experimental parameters constant. The mean size of the obtained PAEC5 (Table 1A and B) by DLS highlights that low Mw of ionic polymer (i.e. <20 kDa) are giving rise to smaller polyelectrolyte complexes while the variation in the number of ionizable groups per macromolecule chain has less significant impact on the mean size of the PAEC5 loaded with ovalbumin.

[0120] Furthermore, PAEC5 were prepared with ovalbumin solutions of concentration ranging from 15 μg/mL up to 1150 μg/mL, while keeping constant the weight ratio between the protein and the ionic polymer (1/2). FIG. 1 shows the evolution of the mean light scattering Intensity (Id cpm) and mean diameter of the PAEC loaded with ovalbumin in function of the concentration of ovalbumin used in the formulation. As outlined on FIG. 1, a linear relationship was observed between the mean light scattering intensity of the PAEC nanodispersions loaded with ovalbumin and its concentration. This linear increase in DLS signal is supporting the efficiency of interaction between the proteinaceous drug and the ionic polymer in an extended range of concentration. The mean size of the PAEC5 remains in the colloidal range.

[0121] The efficiency of drug immobilization and drug loading within PAECs was assessed after separation of the free protein from PAEC with a suitable ultrafiltration device with a membrane cutoff selected to retain the PAEC. The concentration of free ovalbumin in the ultrafiltrate, measured using BCA as protein bioassay, has been estimated to 42%, therefore corresponding to a loading effectiveness of 58%. These two percentages are given with respect to the total amount of ovalbumin added originally in the formulation. Besides, a gel electrophoresis in non-denaturation conditions was performed, with deposition of the samples in the center of the gel disposed horizontally. Adopting this geometry, the PAEC and the free proteins could migrate in opposite direction as a function of their own electrokinetics potential sign and intensity.

[0122] FIG. 2 shows a comparison of the electrophoretic profile of free ovalbumin versus ovalbumin associated with ionic polymers, which are either OB006 (PDMAEMA with a Mw of 10 kDa) or DA002 (PDMAEMA with a Mw of 90 kDa). The electrophoretic profile used gel electrophoresis performed in non-denaturation conditions. The signs + and − which are arranged to the right and left of the vertical arrow indicate the polarity of the electric field, which was applied, while the arrow indicates the location of the sample deposition. After separation, the gel was stained using a silver technique. FIG. 2 highlights the difference in electrophoretic direction. Indeed, when associated to one of the ionic polymers, ovalbumin is migrating in direction of the cathode, while free ovalbumin, used as control, moved in direction of the anode. Knowing that the samples and the gels have been equilibrated in a medium buffered at pH 7.4 before proceeding to the electrophoresis, these results are demonstrating the reversibility of the electrokinetic potential of ovalbumin after complexation with an ionic polymer of formula (I).

Example 2. Preparation of Binary Polyelectrolyte Complex with Human Insulin

[0123] Human insulin is a small globular protein having a Mw of 5808 Da made of 52 amino acid residues distributed in two polypeptide chains, chain A (21 residues) and B (31 residues) which are linked by disulfide bonds. Its isoelectric point is 5.3. Despite its relatively small size, insulin is injected parenterally to diabetic patients on a daily basis in order to reduce the risk linked to the development of microvascular and macrovascular complications, which are one of the main cause of morbidity and mortality associated with this disease. Insulin represents therefore an important proteinaceous drug which could benefit from a nanocarrier allowing to cross biological mucosa and improve the bioavailability.

[0124] Poly (2-dimethylamino) ethyl methacrylate is dissolved in a buffer medium as in Example 1. Insulin dissolution requires first dissolution in an acidic medium, such as acetic acid before proceeding to the progressive neutralisation of the protein solution to achieve a neutral pH. Important precautions should be taken to standardize the conditions of insulin dissolution to avoid any molecular aggregates or either micro- or macroscopic aggregates. Accordingly, the dissolution of this protein should be realized using a device which limits shearing and prevent the introduction of air and foam formation during mixing. In a typical dissolution protocol, insulin is prepared at a final concentration of from 0.1 to at least 10 mg/mL by dissolving the lyophilisated powder in acetic acid solution (0.6M). Placed under rotation, the solution is achieved within a time scale of 10 min at room temperature. Neutralisation of this solution is performed by the progressive addition of a strong base solution, such as NaOH (M) and under a careful control of the pH. When achieving at least pH 6.5 the insulin solution should be transparent, but NaOH should still be added in order to reach pH 7.40. Once arrived at the expected concentration, it is also important to future equilibrate the insulin solution in the same buffer which will be used afterwards to prepare the polyelectrolyte complexes. Final insulin solution should be sterilized by sterile filtration conducted within a laminar flow and can stored for future use at −20° C.

[0125] Once dissolved the insulin and the ionic polymers solutions are mixed within a standard recipe according to the same typical procedure disclosed in example 1. At the laboratory scale, this mixing is performed within a polypropylene tube or any other container of a volume adapted in function of the total volume of PAEC suspension requested. PAEC formation between insulin and the ionic polymer has been verified by DLS monitoring both the increase in mean light scattering intensity and the appearance of an autocorrelation curve. From a noisy autocorrelation curve observed in the presence only of, either of the ionic polymer solution or of the insulin solution, a clear exponential curve is observed quickly after mixing these two macromolecules.

[0126] The size distribution in intensity of the PAEC's have been calculated after deconvolution of the autocorrelation curves and disclosed a mean radius in intensity of PAEC in the nanosize range (Table 2).

[0127] In order to identify the optimal macromolecular features of the ionic polymer allowing to control its association with human insulin, we have assessed the influence of their molecular weight (Mw from 13 kDa to 91.4 kDa) and their charge density keeping constant the concentration of the protein and of the polycation (15 and 30 μg/mL respectively).

[0128] Interestingly, compared to results observed with PAECs loaded with ovalbumin, the Mw and the total number of ionizable groups per macromolecule affects also the mean size of PAECs containing an ionic polymer and insulin but in a different way. Indeed: [0129] a significant evolution of the DLS results is observed between analysis realized 5 and 30 min after mixing the ionic polymer and insulin. These changes of PAEC aggregation with time, not observed with ovalbumin, strongly suggests that the kinetics of the formation of these polyelectrolyte complexes is slower with human insulin; [0130] a decrease of the mean size of the PAEC is observed with a higher molecular weight ionic polymer, a result which contrasts to the opposite evolution noticed with ovalbumin loaded with the same polymers; [0131] the dependence of PAEC formation with the charge density of the ionic polymer is also different compared to results acquired with ovalbumin, the mean size of the nanodispersions decreasing by raising the charge density of the polycations.

TABLE-US-00003 TABLE 2A T 5′ T 30′ Mw Radius at %50 (nm) %50 (nm) 13000  664 +/− 20 5752 +/− 345 26400 1527 +/− 50 730 +/− 34 49100 731 +/− 4 350 +/− 23 91400  300 +/− 35 146 +/− 35

TABLE-US-00004 TABLE 2B T 5′ T 30′ % ionisation Radius at %50 (nm) Radius at %50 (nm) 30 664 +/− 15 5752 +/− 345 47 2753 +/− 205  350 +/− 120 60 847 +/− 22 318 +/− 35 80 847 +/− 25 200 +/− 22

[0132] Table 2A shows the evolution of the mean radius (percentile 50) of PAECs loaded with human insulin in function of the Mw of ionic polymer keeping constant the ionisation % to 30%. Table 2B shows the evolution of the mean radius (percentile 50) of PAECs loaded with human insulin in function of the % of ionic groups to the total number of repetitive units in the ionic polymer adopting a Mw of the ionic polymer of 13 kDa. FIG. 3 shows the evolution of the mean light scattering Intensity (Id cpm) and mean diameter of the PAEC loaded with human insulin in function of the concentration of human insulin used in the formulation, keeping the weight ratio between the protein and the ionic polymer constant at 1:2.

[0133] PAECs were loaded with human insulin in a concentration of from 500 μg/mL up to 5000 μg/m L, while keeping the weight ratio between the protein and the ionic polymer constant at 1:2. FIG. 3 shows graphs which illustrate the evolution of the mean light scattering Intensity (Id cpm) and mean diameter of the PAECs in function of the concentration of human insulin used in the formulation. The left hand y-axis represents a scale for Id in cpm, the right hand y-axis represents a scale for diameter in nanometres, and the x-axis represents a scale for the human insulin concentration in mg per millilitre. As outlined on FIG. 3, an increase in mean light scattering intensity of the PAEC nanodispersions is observed this relationship is however not linear as noticed for ovalbumin. But surprisingly enough this increase of the ionic polymer and protein concentration is linked to a 5 times reduction in the mean size of the nanodispersions. This is advantageous because a decrease in size of PAEC is a benefit to enhance their diffusion within the body and to avoid their capture by immunological systems. This result is surprising because when the concentration of the ionic polymer and of the proteinaceous agent is increased, the probability of particle collision and therefore the risk of aggregation is increased.

[0134] The efficiency of drug immobilization and drug loading within PAECs has been assessed after separation of the free protein from PAEC adopting a suitable ultrafiltration device with a membrane cutoff selected to retain the PAEC. The concentration of free human insulin in the ultrafiltrate, measured using BCA as protein bioassay, has been estimated to 73% when combined with an ionic polymer in a protein to polymer wt ratio of 1:2. This percentage is given with respect to the total amount of human insulin added originally in the formulation.

[0135] The comparison of the electrophoretic profile of free human insulin versus insulin associated with PDMAEMA demonstrated the reversion of the electrokinetic potential of human insulin upon association to this ionic polymer.

[0136] In view to detect some further possible changes in the conformation of the human insulin after its association with the ionic polymer, we have compared its circular dichroism spectra (CD) acquired either on the free form of this protein, or either after its association with 3 ionic polymers. From these CD spectra we have calculated the % of helix in human insulin overtime of storage at 4° C. on 12-day period. This data, illustrated in FIG. 4 and set out in Table 3, highlight that CD spectra of free insulin changes significantly on one-week period whilst after complexation with the polymer A23, the polypeptide gives a rather similar signal. This CD spectra comparison therefore supports that the association of human insulin with ionic polymer provides a stabilisation of the secondary structure of this protein. In particular, FIG. 4 shows CD spectra analysis of either free insulin or insulin associated to an ionic polymer: A23 (PDMAEMA homopolymer with 26.4 kDa); B24 (PDMAEMA homopolymer with 14 kDa with a mean ionisation of 60% after quaternization) or DA002 (PDMAEMA homopolymer with 90 kDa). The CD spectra were taken one day after PAEC preparation.

[0137] Table 3 shows the percentage helix content in human insulin calculated from the ratio ([θ].sub.222/[θ].sub.223) determined from the CD spectra of either free insulin or of insulin after its association with one of these ionic polymers, A23, B24 or DA002. The CD analysis was carried out after storing the PAEC for 1, 6 or 12 days at 4° C.

TABLE-US-00005 TABLE 3 Day 1 Day 6 Day 12 Human Insulin 14.6 37.4 36.9 PECA23p-2A1 22.6 16 19.8 PECB24p-2A1 31.3 23.3 16.9 PECDA002-2A1 26 16.6 21.1

[0138] The morphology of the PAECs loaded with human insulin was also examined by transmission electron microscopy (TEM) (FIG. 5). The images show the nano-range size of these particles as first observed by DLS. More homogeneous and small nanoparticles were produced when the ionic polymer was bearing a poly(ethylene oxide) sequence according to formulae where PEO and PDMAEMA have Mw of 500 and 7000 respectively (FIG. 5B). In particular, FIG. 5A shows TEM images (at 20× magnification) of PAECs loaded with human insulin with PDAEMA without poly(ethylene oxide). FIG. 5B shows TEM images (at 20 000× magnification) of PAECs loaded with human insulin with an ionic polymer was bearing a poly(ethylene oxide) sequence according to formulae where PEO and PDMAEMA have Mw of 500 and 7000 respectively.

Example 3. Preparation of Ternary Polyelectrolyte Complexes with Human Insulin and a Surfactant

[0139] Ternary polyelectrolyte complexes made from human insulin, an ionic polymer referred to as DA002 and surfactant, namely sodium dodecyl sulphate (SDS) were prepared. To a sodium phosphate buffer medium, 333 μL of human insulin solution (6 mg/mL) and 125 μL or 375 μL of SDS (20 mg/ml) were added. Five minutes after addition of SDS, the formation of PAEC was formed by the rapid addition of 1000 μL of the ionic polymer DA002 (10 mg/mL).

[0140] The influence of the molecular weight was assessed (Mw from 10 kDa to 90 kDa) keeping constant the concentration of the protein and of the polymer (2 and 4 mg/mL respectively) and a SDS concentration of 1 mg/mL. The results are shown in Table 4. Interestingly compared to results observed with binary PAECs loaded with human insulin without surfactant, the Mw is affecting the mean size of PAECs in a similar way. Indeed, a decrease of the mean size of the PAEC is observed adopting higher molecular weight ionic polymer, a result which contrasts to the opposite evolution noticed with ovalbumin loaded with the same polymer. Only a slight increase in mean size is noticed 1 day after PAEC preparation and storage at room temperature.

[0141] Table 4 Evolution of the mean radius (percentile 50) of ternary PAEC made from human insulin, SDS and ionic polymer of Mw ranging from 10000 to 90000. DLS mean size have been measured 1 h and 1 day after PAEC preparation and storage at room temperature.

TABLE-US-00006 TABLE 4 T 1 h T 24 h Mw PDMAEMA Radius at %50 (nm) Radius at %50 (nm) 10000  4872 +/− 2850  94863 +/− 11082 20000 206 +/− 8 260 +/− 2  40000 231 +/− 8 260 +/− 26 90000 266 +/− 4 315 +/− 14

Example 4. Preparation of Quaternary Polyelectrolyte Complexes with Vaccine Protein (P24), an Oligonucleotide Adjuvant (CpG) and a Surfactant (SDS)

[0142] P24, a structural protein of HIV viral capside was selected as model biopharmaceutical active for vaccine delivery purpose. This recombinant protein antigenic protein has a Mw around 20 KDa.

[0143] CpG, a short single-stranded synth etic DNA molecule, is well-known to boost the generation of humoral and cellular vaccine-specific immune responses, has been co-immobilized with P24 within PAEC made of one of the ionic polymers corresponding to formula I, for example an homopolymer made from DMAEMA with a Mw of 90 kDa.

[0144] P24 was provided as a 1 mg/mL stock solution and stored at −70° C. After being thawed for at least 15 min at room temperature, this protein was diluted in a sodium phosphate buffer. CpG, under a sodium form, has also been dissolved at 1 mg/mL in a sodium phosphate buffer.

[0145] A surfactant solution, such as SDS, is prepared under a stock solution of 1 mg/mL in sodium phosphate buffer solution. This solution is further diluted in the buffer medium in order to adapt its concentration to the required drug loading.

[0146] Once equilibrated at room temperature, the P24 protein solution and the CpG solution are first mixed with the surfactant solution. Typically, CpG and P24 are mixed in a 1/1 weight ratio, while the ionic polymer is present in a 2 times excess to P24. The final concentration of the surfactant is fixed as a function of the total amount of protein P24 immobilized within the PAEC. This solution is equilibrated for some minutes at 25° C. The ionic polymer is then added.

[0147] PAEC formation between P24, CpG, a surfactant and the ionic polymer has been first verified by DLS. FIG. 6 shows the kinetics of formation of quaternary PAEC made from CpG, P24, SDS and an ionic polymer (DA002) controlling the mean light scattering intensity (Id) and the mean size of the PAEC suspension by DLS. The final concentration of P24 was fixed at 250 μg/mL. The left hand y-axis shows Id (cpm) values for the line with diamond shaped data points and the right hand y-axis shows mean size (nm) values for the line with circular shaped data points.

[0148] As can be seen from FIG. 6, the kinetics of formation is rapid with a maximum in mean light scattering intensity (Id) already noticed 10 min after addition of the ionic polymer. With a P24 concentration of 250 μg/mL, the mean size of the PAEC suspension ranges between 250-270 nm. FIG. 6 shows graphs illustrating the kinetics of formation of quaternary PAEC made from CpG, P24, SDS and an ionic polymer (DA002) controlling the mean light scattering intensity (Id) and the mean size of the PAEC suspension by dynamic light scattering (DLS). The final concentration of P24 was fixed at 250 μg/m L.

[0149] FIG. 7 shows the stability of quaternary PAEC made from CpG, P24, SDS and an ionic polymer (DA002) made with a P24 loading ranging from 50 to 250 μg/mL and stored at 4° C. The stability of the nanodispersions was monitored by DLS, measuring the mean size of the PAEC. FIG. 7 shows a graph illustrating the stability of quaternary PAEC made from CpG, P24, SDS and an ionic polymer (DA002) made with P24 loading ranging from 50 to 250 μg/mL (shown on the x-axis) and stored at 4° C. The stability of the nanodispersions was monitored by DLS, measuring the mean size of the PAEC.

[0150] When stored at 4° C., the PAEC remains stable over a period of at least 12 days considering P24 loading in a range of at least 50 to 250 μg/m L. Surprisingly enough, when raising the concentration of surfactant, up to a critical concentration of 200 μg/ml, the PAEC nanodispersions are homogeneous and stable. Above this concentration, heterogeneous and instable dispersions were noticed. These qualitative observations have been confirmed by DLS with an increase in mean light scattering intensity and a drastic increase in mean size of the PAEC (FIG. 7).

[0151] The comparison of the electrophoretic profile of free P24 associated with ionic polymers, either OB006 (PDMAEMA 10 kDa) or DA002 (PDMAEMA 90 kDa), clearly highlights a difference in electrophoretic direction. Indeed, as disclosed on FIGS. 9 and 10, when associated to these ionic polymers, P24 is migrating in direction of the cathode, while this protein under a free form is moving in direction of the anode. Knowing that the samples and the gels have been equilibrated in a medium buffered at pH 7.4 before proceeding to the electrophoresis, these results are demonstrating the reversion of the electrokinetic potential of P24 when associated to these ionic polymers.

[0152] FIG. 8 shows the mean DLS diameter (measured on the y-axis in units of nm) of 6 batches of PAEC measured one hour after formation, one of them being submitted to 3 repetitive freeze-thaw cycle. The mean size of this batch 6 has not been affected, highlighting the stability of PAEC against this physico-chemical stress. The respective concentrations of P24/CpG/SDS/ionic polymer DA002 were 200/80/200/350 μg/m L. The batch number 5 has also been submitted to 3 freeze-thawing cycles.

[0153] FIG. 9 shows a comparison of the electrophoretic profile of free P24 with P24 associated with cationic polymers, either OB006 or DA002, using gel electrophoresis in non-denaturation conditions. The signs + and − indicate the polarity of the electric field which has been applied, while the arrows refer to the place for sample deposition. After separation, the gel was stained using a silver technique.

[0154] FIG. 10 shows a comparison of the electrophoretic mobility of PAEC loaded with respective concentrations of P24/CpG/SDS/ionic polymer DA002. Their respective concentrations in μg/mL are as follows: 0 (P24)/0 (CpG)/200 (SDS)/0 (ionic polymer) (track 1), 0/0/200/350 (track 2), 50/50/100/250 (track 3), 0/0/100/125 (track 4), 200/80/200/0 (track 5), 50/50/50/0 (track 6), 200/80/200/350 (track 7), 50/50/200/125 (track 8), 50/50/100/125 (track 9), 50/50/50/125 (track 10), 50/50/20/125 (track 11), and 50/50/10/125 (track 12).

[0155] The electrophoretic mobility of the PAEC was analysed using a Coulter Delsa 440SX equipment, working under a constant voltage mode (10 volts) at a temperature of 25° C. The PAEC samples were first equilibrated in a phosphate buffer medium before carrying out the analysis. The evolution of the mean electrophoretic mobility of PAEC prepared according to their compositions, i.e. playing on the SDS concentration, but also on the weight ratio between P24, CpG, SDS and ionic polymers, is shown on FIG. 10. This physico-chemical analysis of the surface properties of the PAEC samples allows the following findings to be drawn: [0156] whatever the composition or the formulation methods, the PAEC always are positively charged. This unexpected observation highlights the predominant contribution of the polycation on the electrokinetic potential of the PAEC and accordingly to its preferential migration to the PAEC surface. Accordingly, we can anticipate that the most significant part of the protein P24, of the oligonucleotide and of SDS have been associated to the ionic polymer and are mostly internalized within the core of the PAEC. Although the % in weight of the ionic polymer is typically greater than the other components of the formulation for all formulations assessed, its preferential migration to the surface of the PAEC is not straightforward. Indeed, both the protein and the surfactant or their combination, could be preferentially concentrated to the surface of PAEC keeping into consideration their well-known surface activity. [0157] a significant decrease in electrophoretic mobility of the PAEC is observed when raising their SDS content, but without reversing the direction of the PAEC mobility in the electrical field. [0158] the control made from a mixture of SDS and the ionic polymer 0/0/10/12.5 is supporting the existence of ionic complexation between this anionic surfactant and this synthetic polymer.

[0159] The fraction of SDS under a free form, i.e. not associated to the PAECs, was determined on the following composition: P24/CpG/SDS/DA002: 200/80/200/350 μg/mL. This analysis was done after separation of the PAECs from free SDS, using ultrafiltration. The free SDS concentration in the permeate fraction was then determined by HPLC using an AZorbax 80A Extend-C18 column. 3.0×150 mm, 3.5 μm from Agilent and an Evaporative Light Scattering Detector (ELSD) Alltech® Model 3300.

[0160] The HPLC analysis indicates that this purification procedure allows to retain more than 99% of the PAEC (190±2 nm in diameter) on the filter, that less than 2.5% of the total amount of the surfactant is free, thus not associated to the polymeric nanostructures retained on the filter and that free surfactant is not adsorbed on the ultrafiltration membrane. According to this analysis, the maximum concentration of free SDS in this PAEC formulation is equal or below 5 μg/mL (Table 5).

TABLE-US-00007 TABLE 5 Particle recovery SDS recovery (Id %) * (wt %) ** PAEC made from 0.06 ± 0.01 <2.5 p24/CpG/SDS/DA002 Free SDS 0.78 ± 0.07 100.0 Mixture of DA002 + SDS 4.79 ± 1.08 <2.5

[0161] Table 5 shows particle and SDS recovery determined in the filtrate fraction of ultrafiltration of PAEC, free SDS or a mixture of SDS and a cationic polymer DA002. Particle recovery has been estimated from the mean light scattering of the permeate fraction of ultrafiltration. The particle recovery percentage was measured as the mean and SD of light scattering count rate of 3 measurements; the SDS recovery percentage was measured as mean and SD of 2 HPLC analysis.

[0162] FIGS. 10A and 10B show TEM micrography (5.000×) of PAECs made from either CpG/P24 (20/20 μg/mL) (FIG. 11A) or CpG/P24 (20/20 μg/mL+SDS 20 μg/mL) (FIG. 11B). FIGS. 11A and 11B illustrate the morphology of the PAECs and confirm the nano-size range of these particles as observed by DLS, although they were mostly under an aggregated state which could originate from the drying required for electron microscopy observation. The images show that more homogeneous and smaller nanoparticles were produced when the ionic polymer was mixed with the surfactant SDS. (FIG. 11B).

[0163] The release rate of the protein P24 and CpG was estimated in vitro in an indirect way, by following the DLS signal of the PAEC suspensions at different temperatures. The comparison of the DLS data summarized in FIG. 13 shows that if the PAEC suspensions are stable at 4 and 20° C., their photocorrelation signal is rapidly lost when they are thermostatized at 37° C. This sharp decrease in mean light scattering intensity correlated with a large increase in polydispersity index of the nanodispersions, the apparition of a noisy autocorrelation curves and without any apparition of sediments in the medium, are strongly supporting the hypothesis of a dissociation of the PAECs when they are incubated at 37° C. The release rate is therefore occurring in the range of the body temperature. In particular, FIG. 13 shows an analysis of the medium term stability of the PAEC loaded with CpG and P24 in a PBS medium at temperatures of 4, 25 or 37° C. The evolution with time of the mean light scattering Intensity has been analyzed up to 90 days.

Example 5. Preparation of Quaternary Polyelectrolyte Complexes with Vaccine Protein (P24), an Oligonucleotide Adjuvant (CpG) and Different Surfactants

[0164] Other surfactants than SDS have been evaluated for their potency to promote formation of PAECs and assure their stability. Following groups were tested: [0165] a family of aliphatic compounds of chemical structure homologous to SDS but differing either in the total number of carbons or in the nature of the anionic group (carboxylic group) [0166] sodium salts of cholic derivates, i.e.: sodium salt of cholic acid, taurochic acid or deoxycholic acid; [0167] non-ionic surfactants: Pluronic PE 6800, Pluronic F127, Tween 20 and Tween 85

[0168] The preparation of these PAECs was performed according to example 1, adopting the respective concentrations of P24/CpG/surfactant/ionic polymer (DA002) of 50/20/2 to 100/90 (μg/mL).

[0169] The overtime monitoring of PAEC formation by DLS analysis in the presence of these different amphiphilic molecules has allowed us to constitute three groups: [0170] Group 1: surfactants which provide formulations with very low count rate (background noise level or close). To this group, belong: —the more hydrophilic surfactants of the carboxylic acid family, i.e.: C.sub.6—COOH and C.sub.8—COOH, carboxylic acid salt with the shorter chain namely C.sub.10—COO.sup.−, Pluronic F127, the cholate family, samples Chol, D-Chol (deoxycholate), and T-Chol (taurocholate); [0171] Group 2: surfactants which provide PAECs whose characteristics improve (size and concentration) with concentration, i.e. higher surfactant concentration enhancing PEG formation. This group include anionic surfactants C.sub.12—COO.sup.− and C.sub.12—SO.sub.4.sup.2−, but also polymeric amphiphilic surfactants such as Pluronic PE6800; and [0172] Group 3: surfactants which favour PAEC formation with better results achieved at lower concentration. This observation has been noticed for the anionic surfactant C.sub.18—SO.sub.4.sup.2− and the neutral surfactants Tween 20 and Tween 85.

[0173] The results are illustrated in FIG. 12 which shows a comparison of the mean light scattering intensity (Id) measured by DLS of PAECs loaded with respective concentrations of P24/CpG/surfactant/polymer were 50/20/2 to 100/90 μg/mL and in function of the surfactant type and of their concentration in the quaternary complexes.

[0174] If the cholate derivatives are excluded from the analysis, the evolution in the property of these surfactants to enhance PAEC formation and to stabilize them is clearly affected by the HLB of these surface proteinaceous agents. Indeed: [0175] below a given aliphatic length of 12 carbons, the anionic alkyl surfactants seem to be too hydrophilic to promote the NP formation. [0176] below a HLB of 14.8, when we reach the critical alkyl chain length of 12 carbons, PAEC formation is promoted, but not with the same extent as observed in the presence of the anionic sulphate binding site present in SDS. [0177] for more hydrophobic compounds which have low CMC, PAEC formation is promoted at the lowest concentration of the surfactants. Indeed, CMC values of C.sub.18—SO.sub.4.sup.2− and of Tween 20 are respectively about 6 times and 100 times lower than SDS. Accordingly, very low concentration of these two surfactants are required to produce stable PAECs.

[0178] All these observations support the hypothesis that PAEC formation and their stability are directly dictated to the CMC and HLB of this surfactant family. Moreover, this systematic study highlights that PAEC formation is also promoted in the presence of a non-ionic surfactant (such as Tween 20, Tween 85 and Pluronic F68). This observation is therefore indicative that PAEC formation can rely only on hydrophobic or hydrogen bondings and not necessarily from ionic interaction between the polycation/protein and the surfactant.

Example 6. Encapsulation of PAECs Loaded with Human Insulin within Alginate Beads

[0179] To prepare an oral composition of PAECs, the PAECs may need to be protected from the gastric environment. To facilitate their transfer and release in the intestine, they have been immobilized in alginate microparticles of a size adaptable between at least 500 to 1000 urn. The PAECs have been first prepared according to previous examples in order to achieve concentration of human insulin of 150 μg/mL, of ionic polymer (DA002) 300 μg/mL, with or without SDS (150 μg/mL).

[0180] The encapsulation of the PAEC suspension in alginate is realized by the following steps: [0181] 1. 0.5 mL of 4 wt % sodium alginate solution is mixed with an equivalent volume of each of the solutions of PAEC; [0182] 2. This mixture is sprayed at a flow-rate of 0.2 mL/min within 25 ml of a 2% CaCl.sub.2) solution; [0183] 3. The Ca-alginate microparticles formed were washed three times by decantation within 0.9% NaCl; and [0184] 4. The microparticles are stored in 2 mL of buffer containing NaN.sub.3 (0.01%) and are stored at 4° C. until future use.

[0185] To assess the efficiency of this formulation step, the morphology of the microparticles was observed under optical transmission (see FIGS. 13A and B). FIG. 14A shows a TEM micrography image (5.000×) of alginate microbeads loaded with binary PAEC. FIG. 14B shows a TEM micrography image (5.000×) of alginate microbeads loaded with ternary PAEC stabilized with SDS. There is a difference of optical aspect between the two alginate beads loaded with two different batches of PAECs. For microparticles loaded with PAECs prepared without SDS, the presence of numerous particles entrapped within the beads are well visible under optical microscopy (FIG. 14A), thus with a size well above their initial mean particle diameter (i.e. 200 nm). In contrast, for microparticles loaded with PAEC stabilized with SDS, no microaggregates are noticed within the alginate beads (FIG. 14A).

[0186] The efficiency of release of the PAECs has also been determined after redissolution of the microbeads in a PBS medium containing EDTA, which is a calcium complexation agent able to trigger the rapid solubilisation of the beads. The DLS analysis of the particles released in the medium shows the release of the PAEC in the medium on a time scale of a few minutes.

Example 7. Preparation of Amphiphilic Electrolyte Complexes (PAEC) Made of CpG/P24/SDS/DA002 in View to Compare their Stability

[0187] Preparation of Amphiphilic Electrolyte Complexes (PAEC) made of CpG/P24/SDS/DA002 with a view to compare their stability on 1 day period according to the following three experimental conditions: [0188] storage at 4° C. under the form of suspension; [0189] storage at −20° C.; and [0190] storage at room temperature after lyophilisation.

[0191] PAEC have been made combining the antigenic protein P24, the oligonucleotide CpG, SDS and the polymer DA002 according to the protocol outlined in Example 4 to produce formulations with a composition identical to those assessed as reported in Example 6.

[0192] The mean size of three batches of PAEC, prepared using the same composition and same procedure has been controlled by DLS just after preparation and after their storage either: [0193] in suspension at 4° C.; or [0194] after freezing imposed after PAEC preparation, storage at −20° C. and thawing 1 day after; or [0195] after a freeze-drying cycle imposed after PAEC preparation, storage at 20° C. and redispersion in an aqueous medium.

[0196] As disclosed on FIG. 15, no significant difference in the mean size of the PAEC can be noticed whatever their mode of conservation. After lyophilisation, these PAEC are instantaneously dispersed using an aqueous phase. Accordingly, these PAEC can be prepared and stored under different physical forms, liquid suspension, frozen state or dried state in function of the final wishes of the end-users.

[0197] FIG. 15 shows a comparison of the mean size of three batches of PAEC made of CpG/P24/SDS/DA002 which have been prepared using the same composition and same procedure as given in Examples 4 and 6 either just after preparation of the PAEC (Initial) and after their storage either: in suspension at 4° C., series labelled “4° C.”; or after freezing imposed after PAEC preparation, storage at −20° C. and thawing 1 day after, series labelled “Freeze-Thawing cycle”; or after a freeze-drying cycle imposed after PAEC preparation, storage at 20° C. and redispersion in an aqueous medium, series labelled “Freeze-Drying cycle”.

Example 8. Nasal Administration of Quaternary Polyelectrolyte Complexes Made from a Vaccine Protein (P24), an Oligonucleotide Adjuvant (CpG) and a Surfactant (SDS)

[0198] The aim of these vivo experiments was to evaluate the immunological response of mice after nasal administration (IN) and intramuscular injection (IM) of quaternary polyelectrolyte complexes made from an ionic polymer (PDMAEMA), a vaccine protein (P24), an oligonucleotide adjuvant (CpG) and a surfactant (SDS), prepared as described in Example 7. The vaccine protein P24 is a proteinaceous agent with a molecular weight of 24 000 Da.

[0199] The immunization and bleeding scheme is as follows: a first injection (IN or IM) at day 0 in C57/B6 mice, followed by a second injection at day 28. The systemic response analysis is done at day 35 (Ag specific cytokine producing T cell frequency) and at day 42 (Ag specific antibody response).

[0200] The experiment was performed in accordance with the Guidelines for animal experiments and using C57/BL6 female mice of approximately 6-8 weeks old. All mice for each experimental group were anesthetized (IP injection: atropine/ketamine/dehydrobenzperidol/fentanyl) and immunized by dropping 10 μl of liquid-vaccine candidate into each nostril or by intramuscular injection. All formulations were injected ex-tempo.

[0201] Each group of 12 mice has been divided into 4 different subgroups of 3 mice for partial bleeding 7 days after second injection (peripheral blood lymphocytes collection for statistical analysis). Samples of serum (12 mice per group) were obtained from mice that were sacrificed 2 weeks after the final immunization. The quantification of the total serum IgG immunoglobulin, total IgA and specific IgG and IgA against the HIV-p24 protein at that time point was all done by ELISA.

[0202] ICS (Intracellular Cytokine Staining) is a flow cytometry-based method and permits not only the detection of cytokine-positive cells, it also allows the identification of the responding cells: Cytokine-producing CD4+ or CD8+ T-cells. Flow-cytometry allows to analyze cytokine-producing cells at a single cell level and to determine “polyfunctional” CD8+ or CD4+ T-cells producing IFNg, TNFa, and IL2 simultaneously. Peripheral blood lymphocytes are stimulated with a pool of peptide encompassing the whole HIV-p24 protein (15-mers peptide overlap by 11) for 2 H after which Brefeldin A is added for the next 16 H. Cells are stained with monoclonal antibodies to CD4-APC Cy7 and CD8-PerCp and after fixation and permeabilization (cytofix/cytoperm from Brefeldin A) for intracellular cytokines (IFNg-APC, IL2-FITC & TNFa-PE). Peptide-specific T cells producing IFNg and/or IL2 and/or TNFa are detected by flow cytometry. Results are expressed as a frequency of cytokine positive cell within CD4 & CD8 T-cells.

[0203] Sera have been analysed for antigen-specific IgG titer (& IgA for nasal wash). 96-well plates have been coated with the antigen at a recommended concentration before sera dispatching plates has been saturated. Consequently, diluted mouse samples have been added and incubated.

[0204] After washes, revelation-step is different for IgG and IgA. For IgG, the plates have been incubated with biotinylated anti-mouse total IgG diluted and 96 w plates have been washed again. After a streptavidin peroxydase (Amersham)/TMB step, the reaction has been stopped. For IgA, the plates have been incubated with diluted peroxydated anti-mouse IgA and 96 w plates have been washed again. After TMB step, the reaction has been stopped. In both cases the absorbance has been read by an Elisa plate reader and results have been calculated using the softmax-pro software.

[0205] The results of the immunological responses noticed for each formulation administrated either by intranasal instillation or by intramuscular injection are reported in FIGS. 16 to 19.

[0206] Formulation controls have been introduced in these experiments in order to better evaluate the immunological potency of the PAECs loaded with the P24 antigen and CpG in the presence of SDS as surfactant. For the nasal administration, these controls are made from the free form of the antigen P24 with free CpG. For the intramuscular injection, free P24-CpG controls has been introduced as a water-in-oil emulsion formulation containing these two biopharmaceutical actives. In all conditions tested by intramuscular injection, an additional adjuvant (AS) has been co-administrated in order to reinforce the immunological response.

[0207] The in vivo results after nasal administration (FIGS. 16A, 16B, and 17) clearly attest of a systemic and specific immunization to the antigenic protein P24 after the administration of PAEC. The systemic immunological response has been analysed monitoring in the blood the CD8+ T-cells producing IFNg, TNFa, and IL2 (FIG. 16A) and CD4+ T-cells producing IFNg, TNFa, and IL2 (FIG. 16B). FIG. 17 shows the systemic immunological response observed on mice after intranasal administration (IN) of quaternary polyelectrolyte complexes made from a vaccine protein (P24), an oligonucleotide adjuvant (CpG) and a surfactant (SDS). The systemic immunological response has been analysed monitoring the antibody P24 concentration in the bloodstream.

[0208] Indeed, at least at the highest dose assessed, there is a clear and significant increase of the CD8 cytokine family and of the antibody against P24 detected in serum collected from mice having received this PAEC. If the CD4 cytokine groups are not significantly modified by the antigen loaded in PAEC, it is worth to mention that their respective concentrations are different between the two experimental series. Compared to P24 administrated under a free form, the serum titer of the antibody is more than 10 times higher with the PAEC formulations. Interestingly enough, there was no significant enhancement in a local mucosal immunization response which has been monitored on nasal wash fluid collection (secretory IgA and cellular immune response (TH1/TC1)).

[0209] Accordingly, we showed an efficient translocation of at least the P24 protein from the nasal mucosa to the systemic bloodstream and under an active conformation. The lack of local immunological response noticed on the nasal site of administration could be explained by the fact that most of the antigen is not released at the port of administration but is mostly transported in other biological compartments of the animals, including the general blood circulation.

[0210] The immunological data collected after intramuscular administration of the PAECs made from CpG and P24 also showed a high enhancement of the immunological response against P24 by comparison either to this free antigen or to its water/emulsion formulation (FIGS. 18A, 18B and 19).

[0211] The systemic immunological response has been analysed by monitoring in the blood the CD8+ T-cells producing IFNg, TNFa, and IL2 (FIG. 18A) and the CD4+ T-cells producing IFNg, TNFa, and IL2 (FIG. 18B). FIG. 19 shows the systemic immunological response observed on mice after intramuscular administration (IM) of quaternary or ternary polyelectrolyte complexes made from a vaccine protein (P24), an oligonucleotide adjuvant (CpG) and optionally a surfactant (SDS) and/or an adjuvant (AS). The systemic immunological response has been analysed monitoring the antibody P24 concentration in the bloodstream.

[0212] Indeed, both CD4 and CD8 markers are up to 10 fold increased upon administration of PAEC with regards to the free antigen or its emulsion formulation. This biological enhancement is also observed when monitoring specific antibody against P24 detected in sera, but to a lesser extent.

[0213] We also compared the immunoresponse given by ternary and quaternary PAEC, each made respectively from P24, surfactant, and an ionic polymer and from P24, surfactant, an ionic polymer, and CpG. Indeed, and surprisingly enough all immunological biomarkers are at least 2 times higher with the ternary PAEC thus devoid of CpG, an oligonucleotide which is typically used to activate the immune response.

Example 9. Preparation of Binary Complexes Made from Human Albumin

[0214] PAEC made from human albumin (HSA) is prepared by physical mixing of aqueous solutions at room temperature. The aqueous solutions are previously filtrated on sterile 0.2 μm filters and the whole formulation procedure is realized within a laminar flow. The proteinaceous drug is human albumin with a molecular weight of 66 kDa, an abundant protein in human plasma, which is negatively charged at physiological pH. The ionic polymer is poly(2-dimethylamino)ethyl methacrylate (PDMAEMA), with a molecular weight from 7 kDa to 91 kDa) dissolved in a sodium phosphate buffer medium equilibrated at a pH of about 7.4. Its concentration is ranging between 0.1 to at least 10 mg/mL, depending on the final drug loading, typically 10 to 50 wt %. HSA is dissolved carefully in the same buffer solution.

[0215] The mixing of the ionic polymer and HSA solutions is performed in a polypropylene tube. The protein solution is typically first added in the appropriate vessel and a given volume of ionic polymer solution is quickly added in one time with a suitable injection device, directly within the solution and not against the wall of the vessel. The volume ratio of protein solution to the ionic polymer solution ranges between 1:1 to at least 100:1 and is adjusted in order to afford a rapid and homogeneous distribution of the ionic polymer within the protein solution. Ionic polymer volume is for example 1/10 of the total volume of PAEC solution.

Example 10. Preparation of Ternary Polyelectrolyte Complexes with Human Insulin and a Surfactant

[0216] Immunoglobulin A (also referred to as IgA) is an antibody that plays a crucial role in the immune function of mucous membranes. Made from 2 identical heavy (H) and 2 identical light (L) chains, its molecular weight is of 140 kDa. Ternary polyelectrolyte complexes made from IgGA, an ionic polymer referred to as DA002 and surfactant, namely sodium dodecyl sulphate (SDS) were prepared. To a sodium phosphate buffer medium, 333 μL of IgGA solution (6 mg/mL) and 125 μL or 375 μL of SDS (20 mg/ml) were added. Five minutes after addition of SDS, the formation of PAEC was formed by the rapid addition of 1000 μl of the ionic polymer DA002 (10 Mg/Ml).

Example 11. Heparin coating of a binary polyelectrolyte complexes made from human albumin

[0217] Surface modification of drug carriers, such as liposomes, polymeric nanoparticles has been reported to reduce their clearance and promote their cellular uptake. Heparin, a hydrophilic polysaccharide is able to provide a steric barrier but can also promote cellular internalisation of the nanovehicles. Accordingly, heparin has been adsorbed to the surface of binary polyelectrolyte complexes made from human albumin and from PDMAEMA having a Mw of 10 kDa, 20 kDa, or 90 kDa whose preparation has been disclosed in Example 9. 10 mg of these PAECs have been dissolved in 16 mL of a phosphate medium buffered at pH 7.4. After 1 h of dissolution carried out at room temperature, a solution of heparin from porcine origin has been added to the PAEC solution in order to achieve a final concentration of heparin between 0.0005 wt/vol % and 0.05 wt/vol %. One hour after equilibration, the formulations have been filtrated on 0.2 μm filter, freeze-dried and resuspended in water.

[0218] Summary of Characteristics

[0219] The examples have shown the following characteristics: [0220] rapid kinetics of PAEC formation (30 min); [0221] the mean size of the PAEC can be from 150-500 nm; [0222] the maximum loading capacity of the at least one proteinaceous agent can be in the range of up to 250 μg/mL for P24 and 80 μg/mL of CpG, but can be raised until 2 mg/mL in the case of human insulin; [0223] the PAEC are stable in vitro at 4° C. for at least 2 weeks; [0224] the proteinaceous agent-containing PAEC are resistant to at least one freeze-thawing cycle and to a drying process, such as lyophilisation or spray-drying; [0225] the release rate of the proteinaceous agent(s) may be a function of the temperature and is significantly enhanced around the body temperature. [0226] an anionic surfactant can be largely associated to the PAEC (more than 97.5 wt %) through physically association, mainly internalized. The presence of a surfactant with amphiphilic properties could facilitate the diffusion of the PAEC through biological barriers by acting as a permeation enhancer; [0227] an optional surfactant may be amphiphilic with an adapted Hydrophilic/Lipophilic Balance to promote the nucleation of PAEC formation. Sodium dodecyl sulphate has proved to be convenient for the nanoencapsulation of human insulin and P24/CpG. Other amphiphilic compounds have proved to be also successful, including non-ionic amphiphilic compounds; [0228] the PAEC have a positive Zeta potential which can be adjusted to some extent by adjusting the content of the anionic excipient; [0229] the conformation state of the immobilized biopharmaceutic drug within the PAEC is not affected; [0230] the PAEC are cytocompatible and hemocompatible; and [0231] the PAEC can be loaded within gastroprotective microparticles.