DESC:FIELD OF THE INVENTION
The present invention relates to the field of acute and chronic wound management and healing. More particularly, it relates to wound healing attributes of placental membrane allograft, dehydrated human amnion-chorion (dAmCh) membrane impregnated with the broad spectrum antimicrobial polymer, Polyhexamethylene Biguanide (PHMB).

BACKGROUND OF THE INVENTION
Placenta-derived membranes are widely used as cellular matrices in the treatment of chronic wounds (Hughes et al, 2016), being immunoprivileged they are ideal for allograft therapies. The structural complexity and presence of growth factors, cytokines and remodeling enzymes supports tissue regeneration and healing (Lim and Koob, 2016). Additionally, chorioamniotic membrane is known to have antimicrobial, anti-analgesic and anti-fibrotic properties (Hoang et al, 2014. Johnson et al, 2017). Single layer allografts of amnion membrane (HA) as well as composite amnion-chorion (HACM) grafts are widely marketed and several patents filed in this regard (US20150110850A1;EP2884844AG). Koob et al (2014) reported a five-fold enhanced efficiency in terms of cytokine profile and immunomodulatory effects in the composite graft, Epifix® (MiMedX, GA) as compared to the amnion graft, Aminofix®(MiMedx, GA). There is a differential distribution of ECM macromolecules in the amnion and chorion, the chorionic trophoblast is usually excluded from grafts due to the relative abundance of metalloproteases and other proinflammatory cytokines (Lei et al, 2016). Variation in tissue processing techniques is reported to have significant impact on preserving bioactivity, therefore PURION® processed HA grafts retained more active components in comparison to other similar products (Koob et al, 2014). Preservation procedures such as cryopreservation and dehydration and sterilization by gamma irradiation affect the physical and functional characteristics of grafts (Paolin et al, 2016). Most of the commercially available PM grafts are dehydrated (Ilic et al, 2017), however cryopreservation (eg.Stravix™, Ociris Therapeutics) retains the structural features of the matrix, viability and response to simulated wound environment (Johnson et al, 2017). Characterization of the Chorioamniotic membrane in terms of its structure, and evaluation of its bioactive components in the context of their functional significance have been well defined (Rennert et al,2013.,Koob et al, 2014.,Lei et al,2016.,McQuilling et al,2018). The antimicrobial property of the membrane was reported by some investigators (Yadav et al, 2017. Palankar et al., 2019).

While most of the studies have used viable/cryopreserved composite membranes, evaluation of antimicrobial activity of the dehydrated AmCh membrane or its extracts is important.

Antiseptics are commonly used as an adjunctive treatment for chronic wounds. Polyhexamethylene Biguanide (PHMB) is a safe, broad spectrum antimicrobial polymer most commonly and effectively used in controlling polyspecies microbial load in wounds (Sowlati-Hashijin et al, 2020). Wound dressing impregnated with PHMB, such as PHMB-Bacterial Nano Cellulose, Nemipore, Protosan etc., have improved efficacy. Moreover, the loading of the antiseptic to the dressing material is achieved by a simple procedure (EP1473047B1; de Mattos et al,2019).

The references D1: US20130289724 A1, D2: US8961617B2, D3: US20120010727A1, and D4: US20120010708A1 discloses about the possible use of an allograft having at least one layer of human amnion and chorion tissues for use as wound cover, allograft in abdominal surgery, allograft in sport injury surgery, and as a replacement cover for muscle sheath, respectively. The cited references are different from the present inventions as (a) the developed product do not contain impregnated PHMB; (b) it is a multilayer, rigid construct which needs assembly of allograft, base sheet, and support layer into wound dressing; (c) it do not have an intact amnion-chorion membrane as they are separated by mechanical stripping; (d) the respective dressing is to be placed inside the boundaries of wound; and (e) it mentions about the application of processed viscous amniotic fluid to surgery site to enhance the healing.

D5: IN2456/CHE/2015A discloses a multi-step method for preparation of placental membrane scaffolds for treating wounds. The process in IN2456/CHE/2015A is highly exhaustive and costly when compared to the present invention, as it involves many stages and different operations such as removal of hyaluronic acid and mucopolysaccharides, enzymatic treatment, collagen crosslinking, and freeze drying. The scaffolds are highly processed and do not retain the original properties of intact amnion-chorion membrane. Also, please note that the synthesis method is completely different in IN2456/CHE/2015A and the present invention.

D6: US20200337904 A1 discloses a multi-step method for preparation of dressing including dehydrated placental tissue for wound cleaning. The process in US20200337904 A1 is also very exhaustive and costly when compared to the present invention, as it involves many stages and different operations such as preparation of dehydrated human amnion or chorion membrane, collagen swelling, blending of oxidized regenerated cellulose (ORC), add plasticizer etc. The material is highly processed and do not retain the original properties of intact amnion-chorion membrane. The dressing material contains 2 different layers with added collage, ORC, and plasticizers. Also, please note that the synthesis method is completely different in US20200337904 A1 and the present invention.

D7: “Effectiveness of polyhexamethylene biguanide impregnated dressing in wound healing: a systematic review protocol” JBI Database of Systematic Reviews and Implementation Reports, 2016, summarizes the review of work done in PHMB impregnated dressing for wound healing. The review papers dealt exclusively with healing properties of PHMB compared with silver, iodine, standard dressing, regular dressing, and other antimicrobial dressings. Furthermore, it did not disclose any information regarding the use of human amnion-chorion membrane as in the present invention. Hence, kindly consider that our work is inventive in view of the prior art.

D8: Polyhexamethylene Biguanide: Polyurethane Blend Nanofibrous Membranes forWound Infection Control, Polymers 2019, 11, 915; doi:10.3390/polym11050915, 22 May 2019, discloses the use of nano fibrous membranes prepared by blending PHMB and polyurethane to gradually release PHMB for wound infection control. The synthesis procedure and working principle of the prior art is completely different from the present invention. This results in a product which is in nanoscale regime. Also, the prior art did not disclose any information regarding the use of human amnion-chorion membrane as in the present invention. Hence, kindly consider that our work is inventive in view of the prior art.

In summary, D1 to D4 discloses about development of rigid multilayer wound dressing constructs to be placed inside the boundaries of wounds. D5 and D6 uses highly exhaustive steps to process the amnion and chorion membrane into multilayered material. Also, in the works D1 to D6 the amnion and chorion layers are separated and there was no mention of impregnation of PHMB which makes them entirely different from the present invention. Reference D7 is a review paper that compares PHMB with other competitive materials. Reference D8 is a physicochemical work which discloses the use of nano fibrous membranes. There is no mention about human amnion-chorion membrane in D7 and D8.

The present invention resulted in a new product (PHMB impregnated intact dAmCh), involves technical advance (impregnation of PHMB), have economic significance (minimal processing, minimal manipulation, easy to use, less medical intervention, bio absorbable and hence savings in disposal), and efficacy (synergetic effect of impregnated PHMB and dAmCh membrane in effective wound healing).

The therapeutic possibilities in a combination of PHMB and placental membrane allografts has not been explored and is expected to have a cumulative effect in clearing infectious organisms from the wound.

OBJECT OF THE INVENTION
It is primary object of the present invention to provide a synergistically effective minimally processed, intact dehydrated human amnion chorion membrane (dAmCh) and impregnated PHMB.

It is another object of the present invention to provide a wound dressing material which offers excellent wound healing properties.

It is another object of the present invention to provide a single layer, ready to use wound cover and its one step application makes it user friendly.

It is another object of the present invention to develop product that is bio-absorbable and hence not much medical intervention is required.

SUMMARY OF THE INVENTION
One or more of the problems of the conventional prior arts may be overcome by various embodiments of the present invention.

It is another aspect of the present invention to provide a placental membrane based wound dressing material, essentially consisting of:
a polyhexamethylene biguanide impregnated dehydrated amnion-chorion membrane;
wherein 0.5-2 % polyhexamethylene is distributed in 1 gm dehydrated amnion-chorion membrane.

It is another aspect of the present invention to provide the placental membrane based wound dressing material, wherein 1gm of amnion-chorion membrane comprises of 5 mg of PHMB.

It is another aspect of the present invention to provide the placental membrane based wound dressing material, wherein the dehydrated amnion-chorion is derived from human placental layers.

It is another aspect of the present invention to provide the placental membrane based wound dressing material, wherein the PHMB impregnated dehydrated amnion-chorion membrane has augmented antimicrobial activity and functional efficacy for the treatment of chronic wound.

It is another aspect of the present invention to provide the placental membrane based wound dressing material, wherein the PHMB is composed of repeating basic biguanidine units connected by hexamethylene hydrocarbon chains, providing a cationic and amphipatic structure.

It is another aspect of the present invention to provide the placental membrane based wound dressing material, wherein the antimicrobial agent is PHMB in combination with one or more of Tetracycline, Penicillin, Neomycin, Erythromycin, Clindamycin, Silver, Iodine, Chlorhexidine, Oflaxacin, Ethyl alcohol, Povidine iodine, Framycetin, Mupirocin, Dicloxacillin, Cephalosporins.

It is another aspect of the present invention to provide a method of preparation named AGNES technology where placental membrane based wound dressing material, comprising of steps:
collection of placental tissue;
subjecting the membranes to microbial and infectious disease screening;
washing in sterile water and antibiotic solution;

cleaning the placental tissue with sterile saline;
gentle washing the placental tissue in placental washing solution (2X RBC Lysis solution and 18% NaCl) in 100 rpm shaking incubator for 45-60 minutes;
washing in sterile distilled water in 100 rpm shaking incubator for 10 – 15 minutes;
spreading the membrane on delrin block and keeping the membrane in tyvek pouch for drying at 40 ± 2 degree Celsius in hot air oven for 16-24 hours;
incorporating 0.5% PHMB in AMCH membrane and drying at 40 ± 2 degree Celsius in hot air oven for 16-24 hours; and
slicing the membranes to specific dimensions, sealing and subjecting to sterilization by gamma radiation.

It is another aspect of the present invention to provide the method of preparation of placental membrane based wound dressing material, wherein the slicing of membrane is done using laser.

BRIEF DESCRIPTION OF DRAWINGS
Figure 1 Illustrates the flow chart of method of preparation of AmchoPlast – PHMB membrane.
Figure 2 illustrates the flow chart amnion-chorion membrane with 0.5% PHMB membrane – according to an embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the field of chronic wound management and healing. More particularly, AmchoPlast-PHMB: augmented antimicrobial activity and functional efficacy for the treatment of wounds.

The present invention gives a Minimally processed one-layer flexible membrane with intact amnion and chorion membrane having its regenerative properties. The wound dressing material of the present invention can be placed over the wound or inside the wound also.

Wound healing attributes of placental membrane allograft dehydrated human amnion-chorion (dAmCh) membrane is enhanced by impregnation with the broad-spectrum antimicrobial polymer Polyhexamethylene Biguanide (PHMB). The components in the biomaterial have a synergic effect as evidenced by the augmented antimicrobial effect on planktonic culture of microbial species predominating the polyspecies-biofilms of infected recalcitrant wounds. PHMB-AmchoPlast as an effective anti-biofilm agent would be established further using an in vitro biofilm model of chronic wound.

dAmCh retains the structural and biological properties of placental membrane. The antimicrobial components and the array of cytokines, chemokines and growth factors facilitate repair and regeneration of infected wounds. However, prolonged infection, high microbial load and the temporal dynamics of the wound microbiome can outcompete the antimicrobial potential of the membrane, further making the environment less conducive for reparative agents (Bowler et al, 2001., Lei et al, 2016., Loesche et al, 2017).This is more apparent with biofilm forming species that are resistant to antimicrobial agents; imbalanced production of proteases also degrades growth factors and cytokines impairing the healing process (Mir et al, 2018)

PHMB and dAmCh can have a synergic effect in bringing down microbial load, possibly destabilizing the microbiota in the wound facilitating continuous healing. It is important in a scenario where the wound microbiome can possibly limit the therapeutic potential of the placental membrane.

General overview of the present invention:
• Dehydrated amnion chorion (dAmCh) with augmented antimicrobial efficiency
• Antiseptic PHMB incorporated in dAmCh
• dAmCh retains its wound healing and regenerative properties
• Polyspecies biofilm forming microbial species, in vitro studies
• Synergic antimicrobial effect of dAmCh-PHMB membrane
• Wound more conducive for the therapeutic active components of dAmCh.

According to the present invention, the method of preparation of wound dressing material involves:
The placental membrane collected in aseptic conditions and subjected to microbial and infectious disease screening is used for production of the dressing material. The membrane with intact amnion and chorion layers is gently washed, dried and incubated for a standardized time in a defined concentration of PHMB solution.

Summarized Procedure:
Clean the placental membrane with Purified water (Milli-Q) / normal saline then clean with Placental washing solution (RBC lysis solution containing 18% NaCl) to remove blood stains/extra layers and dehydrate at 40 ± 2°C and incorporate amnion chorion membrane with PHMB and again dehydrated at 40 ± 2°C. Dehydrated PHMB allografts are sterilized and packed.

Preparation of PHMB solution:
0.5% PHMB membrane preparation (w/w):
0.5 g of PHMB in 100 ml of water – 0.5%
0.5 g of PHMB in 100 g of tissue weight – 0.5%
Therefore 500 mg in 100000 mg of tissue.

PHMB concentration requirement in the wound dressing material is 0.5% w/w.

0.125% working PHMB solution preparation:
0.125 gm of PHMB powder shall be dissolved in 100 ml of sterile water for injection.

For e.g., considering the dehydrated membrane weight is 1 gm, it is required to add 4ml of 0.125% PHMB solution to achieve 0.5% of PHMB concentration in Final product (1 gm of tissue contains 5 mg of PHMB).

The dried membrane weight is noted, and soaking volume is calculated based on the weight (WEIGHT*4) of 1.25mg/ml PHMB for 4 hours in the shaker incubator at 100 (+/- 10) RPM. After 4 hours incubation the soaked membrane is removed from the container and spread on the Inverse “LC” Delrin block, where amnion is facing downward and chorion facing upward. Chorionic layer is thicker compared to the amnion layer. The amnion layer is comparatively translucent to the chorionic layer. Delrin block placed in Tyvek pouch should be sealed and incubated in Hot air oven at 40±2 °C for 16-24 hours.

Packaging procedure for subjecting dehydrated membrane to Gamma radiation:
The dehydrated membrane is packed in Primary Pouch and sealed with nitrogen using a vacuum sealer.
Then, primary packaged membrane is kept inside the Pre labelled Secondary pouch and sealed by purging with nitrogen.

After dehydration the membrane is cut using laser with membrane sealed in Tyvek pouch to specific dimensions, sealed and sterilized by gamma irradiation 25kGy.

The physical and functional characteristics of the product are validated by established protocols. In terms of clarity, thickness, moisture content, tensile strength, total protein content, as well as in the presence of cytokines, growth factors, angiogenic and cell proliferative potential, the product is confirmed to be ideally retaining the known properties of dAmCh membrane. The incorporation of PHMB did not alter any of its inherent qualities.

The properties of the Amnion-chorion wound dressing material according to the present invention:
Values are as Mean + Standard Deviation (n=8)
Tensile strength - 4.62 ± 2.81 (Newton)
Moisture content - 9.39 ± 2.80 %
Total protein content - 0.98 ± 0.26 mg/cm2
Thickness - 82.83 ± 16.72 µ
Transparency - 83.14 ± 3.13 µ.

Antimicrobial property: In vitro assays proving the synergic effect of PHMB and dAmCH. A comparison of the inherent antimicrobial properties of dAmCh (AmchoPlast) and dAmCh-PHMB (AmchoPlast-PHMB) in planktonic culture involving microbial species frequently found in a chronic wound microbiome. The efficiency of AmchoPlast-PHMB would be further established by an in vitro-biofilm model of chronic wound.

Synergic effect of antimicrobial components of dAmCh and PHMB in decreasing high microbial load, facilitating the continuation of normal wound healing.

The wound environment can be more conducive to the anti-inflammatory and growth promoting effects of dAmCh in case of recalcitrant chronic wounds.

The amniotic membrane is part of the inner layer of the placenta, this is a bilayer membrane composed of amnion and chorion. Thus, the amniotic membrane if a formidable natural biological barrier that separates the fetal and maternal tissues. It is an immune-privileged tissue. The use of dehydrated placental membranes in wound healing care has been successful in clinical trials. Nevertheless, bacterial infection of the wound remains as a major concern affecting the wound healing process. Hence, coating of the dehydrated amnion chorion membrane with antimicrobial compounds could be an effective strategy to overcome this barrier.

The broad-spectrum antimicrobial Biocide Poly hexamethylene biguanide (PHMB) kills bacteria, fungi, parasites and certain viruses with a high therapeutic index.
,CLAIMS:WE CLAIM:
1. A placental membrane based wound dressing material, essentially consisting of:
a polyhexamethylene biguanide impregnated dehydrated amnion-chorion membrane,
wherein 0.5-2 % polyhexamethylene is distributed in 1 gm dehydrated amnion-chorion membrane.

2. The placental membrane based wound dressing material as claimed in claim 1, wherein 1gm of amnion-chorion membrane comprises of 5 mg of PHMB.

3. The placental membrane based wound dressing material as claimed in claim 1, wherein the dehydrated amnion-chorion is derived from human placental layers.

4. The placental membrane based wound dressing material as claimed in claim 1, wherein the PHMB impregnated dehydrated amnion-chorion membrane has augmented antimicrobial activity and functional efficacy for the treatment of chronic wound.

5. The placental membrane based wound dressing material as claimed in claim 1, wherein the PHMB is composed of repeating basic biguanidine units connected by hexamethylene hydrocarbon chains, providing a cationic and amphipatic structure.

6. The placental membrane based wound dressing material as claimed in claim 1, wherein the antimicrobial agent is PHMB in combination with one or more of Tetracycline, Penicillin, Neomycin, Erythromycin, Clindamycin, Silver, Iodine, Chlorhexidine, Oflaxacin, Ethyl alcohol, Povidine iodine, Framycetin, Mupirocin, Dicloxacillin, Cephalosporins.

7. A method of preparation named AGNES technology where placental membrane based wound dressing material, comprising of steps:
collection of placental tissue;
subjecting the membranes to microbial and infectious disease screening;
washing in sterile water and Antibiotic solution;
cleaning the placental tissue with sterile saline;
gentle washing the placental tissue in placental washing solution (2X RBC Lysis solution and 18% NaCl) in 100 rpm shaking incubator for 45-60 minutes;
washing in sterile distilled water in 100 rpm shaking incubator for 10 – 15 minutes;
spreading the membrane on delrin block and keeping the membrane in tyvek pouch for drying at 40 ± 2 degree Celsius in hot air oven for 16-24 hours;
incorporating 0.5% PHMB in AMCH membrane and drying at 40 ± 2 degree Celsius in hot air oven for 16-24 hours; and
slicing the membranes to specific dimensions, sealing and subjecting to sterilization by gamma radiation.

8. The method of preparation of placental membrane based wound dressing material as claimed in claim 7, wherein the slicing of membrane is done using laser.