Field of the invention

The present invention relates to biodegradable substrates. More specifically the present invention relates to paper-based substrates impregnated with copper based anti-microbial actives.

Background and the prior art
Anti-microbial properties of copper on account of its unique electronic configuration has been widely known. Active copper ions directly interact with genetic material of bacteria, viruses through its oxidative property thereby providing a very wide spectrum of activity.
In the wake of pandemics, there is an unprecedented demand for a variety of apparels and articles that provide surface anti-microbial and anti-viral properties thereby dramatically reducing the chances of contamination and spread of infection. In view of the growing environmental concerns, there is a growing focus on use biodegradable disposables that exhibit surface antimicrobial activity.
One of the most versatile biodegradable substrates that can be used for the manufacture of various disposable apparels and articles with surface anti-microbial activity is paper. However, imparting effective surface anti-microbial properties on paper poses several challenges.
Various types paper substrates with anti-microbial actives, particularly copper, have been disclosed in CN107916595, US9611153, WO2017216602 and CN108532366. However, these products mainly rely on the anti-microbial actives being completely absorbed in the paper substrates unlike being surface adsorbed which ensures higher amounts of actives being accessible for its activity.
Further, active copper ions being extremely susceptible to oxidation upon exposure, retaining them in active states on paper surface is extremely critical to ensure maximal antimicrobial and antiviral activity. Therefore, there exists a need for paper-based substrates impregnated with copper based anti-microbial actives that avoids undue loss of antimicrobial/antiviral activity of copper on account of premature exposure of copper ions on surface to atmosphere.

Object of the invention
It is an object of the present invention to provide biodegradable substrate which has surface antimicrobial properties.
It is another object of the present invention to provide paper-based substrates impregnated with copper based anti-microbial actives with improved microbial kill with long shelf-life.

Brief description of accompanying figures
Figure 1 illustrates the process of preparing the biodegradable substrate of the present invention.
Figure 2 illustrates the growth of E. Coli and S. aureus on the biodegradable substrate of the present invention.
Figure 3 illustrates the cytotoxicity of the drug in MDCK cells as determined by MTT assay.
Figure 4 illustrates the percentage (%) of MDCK cell protection in vitro against H1N1 influenza virus.
Figure 5 illustrates the indirect immunofluorescence assay showing the antiviral efficacy of test compound in MDCK cells against H1N1 virus using an anti-NP antibody.

Summary of the invention
According to one aspect of the present invention there is provided a biodegradable, antimicrobial substrate comprising (i) a cellulose substrate and (ii) a copper nanoparticle colloid comprising 0.22 % to 0.75 % by weight a copper II salt, 1.5 % to 4.5 % by weight a capping agent, 0.1 % to 0.3 % by weight a complexing agent, 0.1 % to 0.35 % by weight a reducing agent and 94 % to 98 % by weight a solvent; said nanoparticle colloid being surface absorbed on to the cellulose substrate.
According to another aspect of the present invention there is provided a process for preparing a biodegradable, antimicrobial cellulose substrate comprising the steps of :
(i) preparing a copper nanoparticle colloid comprising 0.22 % to 0.75 % by weight copper II salt, 1.5 % to 4.5 % by weight capping agent, 0.1 % to 0.3 % by weight complexing agent, 0.1 % to 0.35 % by weight reducing agent and 94 % to 98 % by weight solvent;
(ii) doping a cellulose substrate with the copper nanoparticle colloid of step (i) to provide a thin film on the surface of the cellulose by film transfer method;
(iii) passing the doped cellulose substrate of step (ii) with significant wet tensile property of 0.3 to 0.5 kg/cm through a series of rotating driers under stretch of 1.2 to 1.6% and temperature 70° to 90°C to obtain said biodegradable, antimicrobial cellulose substrate.

Detailed Description of the invention
The present disclosure provides a biodegradable cellulose substrate impregnated with copper based anti-microbial actives. The substrate of the present disclosure exhibits antimicrobial property of elemental copper where the copper is uniformly present on the substrate thereby increasing its surface area by many folds so that the substrate possesses strong antimicrobial property log kill of at least 3 with long shelf life of atleast 4 years.
The cellulose substrate used in the present disclosure is preferably a paper substrate which is completely biodegradable.
The present inventors have surprisingly found that a copper nanoparticle colloid comprising 0.22 % to 0.75 % by weight copper II salt, 1.5 % to 4.5 % by weight capping agent, 0.1 % to 0.3 % by weight complexing agent, 0.1 % to 0.35 % by weight reducing agent and 94 % to 98 % by weight solvent; when impregnated on to a paper substrate provides excellent antimicrobial property. The particle size of the nanocolloid is of the order of 1-1000nm.
The copper II salt used in the present invention is preferably copper sulphate (II) salt.
The dispersing/capping agent used in the present invention is selected from amylose, amylopectin or mixtures thereof. It has been found that the combination of amylose and amylopectin is the most effective. The preferred ratio of amylose and amylopectin is 17:83. Amylose and amylopectin can be same may be natural or synthetic.
The present inventors have found that the nano colloidal solution of the present invention hides the highly reactive reduced copper in its nano state by encapsulating nano particles by amylose- amylopectin, prohibiting them from agglomeration and thus keep them well dispersed in the medium and prevent them from aerial oxidation. The present inventors had tried using the same with acrylic base binder ranging from 0.5% to 3% in place of amylose - amylopectin, however could not get the copper in its reduced copper oxide state, but always got the copper in its metallic state.
The complexing agent used in the present invention is preferably caustic soda.
The reducing agent used in the present invention is selected from sodium borohydride, glucose, hydrazine hydrate, ascorbic acid, citric acid, formic acid or mixtures thereof.
The present inventors have found that fresh water can be advantageously used as the solvent thereby making the process green and cost effective.
The paper substrate impregnated with copper nano-particle colloid in accordance with the present invention is characterized by specific log kill values for viruses and bacteria.
It has been found that the capping agent also acts as a stabilizer and encapsulates the reduced copper ion thereby avoiding undue oxidation of the reduced copper ion on the surface. Copper ion gets oxidized only when it comes in contact with moisture and microbes thereby exhibiting high surface antimicrobial and anti-viral activity.
The present inventors have found that lowering the proportion of capping agent causes poor dispersion due to agglomeration of nano particles, while beyond the level adversely affect the paper functional property for its end use.
Without being bound by theory, it is believed that reduced copper ion in encapsulated state is protected from areal oxidation and while in highly dispersed nano state, applied on paper surface, paper is coated uniformly and in consequence increase its surface contact probability by many fold.
The amount of adsorbed copper on the biodegradable, antimicrobial cellulose substrate of the present invention ranges from 0.045 - 0.15 gm per m2 of paper, preferably 0.045 gm per m2 of paper.
According to a preferred embodiment of the present invention the substrate of the present invention has copper nanoparticle colloid adsorbed to its surface wherein the copper nanoparticle colloid comprises of amylose-amylopectin, Caustic Soda, Hydrated Copper Sulphate, Vitamin C in the ratio 3.3: 0.16: 0.5: 0.25. Preferably when a solution having about. 1250 ppm reduced Copper ion in its nano colloidal state is impregnated on to the paper surface it provides a coating of about 0.045 gm reduced copper per m2 of paper. The copper nanoparticle colloidal solution has the particle size in the range from 1nm to 1000nm, preferably 50-400 nm.
It has been found that the above combination is based on stoichiometric proportion for the controlled chemical reaction in between copper salt and ascorbic acid in desired alkaline medium. The higher copper with lower ascorbic acid, makes the reduction incomplete defeating the purpose of having the total copper content in its reduced state as desired. On the contrary, the higher ascorbic acid with lower copper salt leads to uncontrolled reduction resulting the formation of metallic copper from copper salt defeating the purpose of obtaining a homogenous colloidal solution of reduced copper oxide. Further excess of ascorbic acid makes the resultant solution acidic which has adverse impact on paper properties.
According to the present invention the capping agent imparts the functional ability of the solution, which is selected from amylose, amylopectin and mixtures thereof. It is selected aiming the target for making this particular product to adhere the desired property on its surface by Film Transfer Methodology and taking into consideration of the consecutive processing of the doped wet paper in a series of driers and calendar section of a paper machine.
It has been found that higher concentration of amylose, amylopectin imparts higher viscosity in the medium, which ultimately bring undesired paper property, misfitting for its end use. e.g., water repellence, paper stiffness. Also, lower concentration of amylose, amylopectin results in uncontrolled growth of Nano particles in dearth of particle growth terminator. The reaction will take place but the particles grow freely in all direction. In consequence the particles in the finished product do not look like similar in shape and size due to nonuniform particle clusters owing to agglomeration.
According to another aspect of the present invention there is provided a process for preparing the paper substrate impregnated with copper based anti-microbial actives comprising the steps of :
(i) preparing a copper nanoparticle collioid comprising 0.22 % to 0.75 % by weight copper II salt, 1.5 % to 4.5 % by weight capping agent, 0.1 % to 0.3 % by weight complexing agent, 0.1 % to 0.35 % by weight reducing agent and 94 % to 98 % by weight solvent;
(ii) doping paper substrate with the copper nanoparticle colloid of step (i) for certain duration to provide a thin film on the surface of the cellulose by film transfer method;
(iii) passing the doped paper of step (ii) with significant wet tensile property of 0.3 to 0.5 kg/cm through a series of rotating driers under stretch of 1.2 to 1.6% and temperature 70° to 90°C to obtain the substrate of the present invention.
Figure 1 illustrates the process of preparation of the substrate of the present invention.
The substrate used in the present invention is preferably selected from cellulose having critical absorbency property of 20 min Klemm, definite degree schopper of 30 to 32 and Fiber Length Index of 0.08 to 0.12.
The time duration for doping the paper substrate in step (ii) is 5 to 8 seconds.
Accelerated Aging test of the substrate of the present invention was done and no change in shelf life was observed. The product retains all its properties for minimum 4 years in keeping in undamaged condition.
The paper substrate impregnated with copper nano-particle colloid in accordance with the present invention can be made into apparel and article. The present invention thus finds applications in hospitals or any health care center, for example, for use as bed cover in train berth, as table cover in hotels, restaurant, or as wrapper in salon, take away bags and other packaging applications. The product of the present invention is completely biodegradable and can be produced at a very low cost. The present invention can be applied to all types of cellulose-based packaging materials which include very sensitive courier services or any type of home delivered item under packing.
The present invention is now illustrated by way of non-limiting examples.

Example 1
Process Steps for the preparation of the biodegradable substrate of the present invention:
1/ Making a 50 g/L solution of amylose – amylopectin with filtered fresh water under stirring for 15 minutes. (100 kg amylose – amylopectin in 2000 liter filtered fresh water)
2/ Raising the temperature slowly up to 90 °C for 30 minutes up to raised volume 2500 liter with steam condensate.
3/ Adding 38 g/L copper solution from hydrated copper salt for 10 minutes. (15 kg Pentahydrate Copper Sulphate, commercial grade)
4/ Adding 50 g/L caustic solution for 10 minutes. (5 kg Caustic Soda)
5/ Addition of 25 g/L ascorbic acid solution for 25 minutes (7.5 kg Ascorbic acid)
The below table gives in details the composition of the inputs ingredients.
Table 1
Components Amounts
Amylose-amylopectin 100 kg
Pentahydrate Copper sulphate 15 kg
Caustic soda 5 kg
Ascorbic acid 7.5 kg

6/ The final volume made up to 3000litres. Finally achieving a strictly colloidal solution of reduced copper oxide with a concentration having 1250 ppm copper in it, pH 4.5 to 5.5 and viscosity 3.5 to 4.5 cps at room temperature ( 25 ° C ).
[15 kg Copper Sulphate Pentahydrate having purity 98 % considered,
Input = 14.7 kg
Molecular Weight of pentahydrate copper sulphate = 63.5 + 32 + 64 + 90 = 249.5
% of Cu in pentahydrate salt = 63.5 / 249.5 = 25.45 %
Total input of Cu from 14.7 kg pentahydrate copper salt is = 3.741 kg = 3741 gm
3741 gm copper is in approx. 3000liter final volume to get a solution of 1247 ppm copper concentration, i.e., approximately 1250 ppm Copper.]
Ultimate Components in Final solution of 3000 liter is mentioned below:

Components Amounts
Amylose-amylopectin 100 kg
Reduced Copper Oxide 4.3 Kg
Sodium Sulphate 8.88 kg
Dehydro Ascorbic acid 7.33 Kg

7/ impregnating a paper substrate with the solution having approx. 1250 ppm reduced Copper ion in its nano colloidal state to provide a coating of approx. 0.045 gm reduced copper per m2 of paper copper nanoparticle colloid of step (6) to provide a thin film on the surface of the cellulose by film transfer method.
8/ passing the doped cellulose substrate of step (7) with significant wet tensile property of 0.3 kg/cm ( min ) through a series of 9 rotating driers under stretch of 1.2 % (min ) and temperature 70°C to 90°C to obtain the biodegradable, antimicrobial cellulose substrate of the present invention.
The composition of the colloidal solution is provided below :
Amylose-amylopectin, Caustic Soda, Pentahydrate Copper Sulphate, Vitamin C are in the ratio 3.3 : 0.16 : 0.5 : 0.25
The particle size of the nano colloid was found to be in the order of conforming colloidal character (diameter ranging from 50-400 nm)

Example 2
Evaluation of Antimicrobial activity of porous surface of paper
The bacterial counts on the substrate (paper) of the present invention were lower than detectable limit indicating that coated
paper killed bacterial cells (Matrix - 1)
The bacterial killing activity was observed against both E. coli and S. aureus.
Methodology:
The method of ASSAY applied: Standard MTS based cell survival assay (Protocols: ISO 19007) and that disclosed in United States Patent by Schwartz et al. US008748174B2 US 8,748,174 B2 Jun. 10,2014
E. coli and S. aureus. No growth even after 24 hrs (Figure 2)
Matrix 1: Bacterial counts on Paper

Results and Interpretation: (Six-time repeatability checked)
1. The Paper exhibited antibacterial activity, killing both E. coli and S. aureus over 24-hour contact period.
Final remarks: Growth of Bacteria are below detectable limit( log kill of at least 3)

Example 3
Stability Experiments were performed on the formulation of the present invention disclosed in Example 1 using the Standard MTS based cell survival assay (Protocols: ISO 19007) and that disclosed in United States Patent by Schwartz et al. US008748174B2 US 8,748,174 B2 Jun. 10,2014
Data concerning shelf-life of surface antimicrobial properties of the paper.
Humidity Temperature
deg C Remarks
50% 90, 100 Humidity 50-95% at normal temperature or room temperature does not have any impact in terms of functional as well as microbial properties. Exposure time was 48 & 72 hrs. But continuous exposed to rain may have reduced antimicrobial properties due to surface change.
Accelerated aging test at different temperature 90-150°C does not show any deterioration in microbial activity.
LCA done on log scale found to be 5 years.
70% 110, 120, 150
90%
95%

Example 4
Determination of IC50 values of the test compound, Assessment of in-vitro antiviral efficacy of test compound against Influenza virus (H1N1) and detection of viral nuclear protein in the cells infected with influenza virus by immuno-fluorescence microscopy.

1. TCID50 determination of virus (Influenza)
The TCID50 was determined in replicate cultures of serial dilutions of the virus sample. The
titer of the virus stock was expressed as the TCID50/ml and was calculated using a standard
protocol established in our lab after minor modification
a) MDCK cells were seeded (1.5 x 104) in a 96-well plate and allowed to grow for 24 hours at 37 °C, 5% CO2 under humid environment till the cells became confluent.
b) Next day, cells were washed with PBS and prepared for infection with stock virus.
c) A serial dilution of virus stock starting from at 1:10 in viral growth media (DMEM+1% BSA+P/S+ 1µg/ml TPCK) treated trypsin was added (100/200 µl) each dilution in at least 6 replicate wells and allowed to incubate for 2 hr at 37 0C under 5% CO2 pressure. Appropriate negative control wells were included for only media (no virus infection).
d) After 2 hr, media containing the virus was removed, washed the cells with PBS once and replenished the wells with fresh viral growth media and allow to rest at 37 0C with 5% CO2 for 48-72 hr.
e) After the required time, the plates were placed under microscope and noted down the changes in positive and negative wells (+ve wells will show extensive CPE, while –ve wells will have less than 50% CPE, eye estimation as compared to the condition of the cells in the wells that had no virus infection)

1. Following TCID50 value was calculated as described below
• Calculate Proportionate Distance (PD): (% next above 50%)- 50% / (% next above 50%) – (% next below 50%)
In the example above: PD= 80%-50% / 80%-0%= 30/80= 0.375
• Now calculate 50 % end point dilution as
Negative logarithm of TCID50 titre = (negative logarithm of the next dilution above 50% mortality + PD) x log dilution factor
In the Example above:
negative logarithm of the next dilution above 50% mortality = -6
PD = 0.375
Dilution factor = log(10) i.e. 1
Thus, negative logarithm of TCID50 titre is (-6+0.375)x1 = -5.625
Hence, log TCID50 titre is 10-5.625 i.e. 2.37x10-6 or 1/ 2.37x106
• Now Calculate TCID50/ml as (1/ log TCID50) / the ml of viral inoculum
added
In the above case:
TCID50/ml= 2.37x106/ 0.2 = 1.18x107/ml

2. IC50 Determination of the compound coated onto the paper
a) MDCK cells were revived in a T25 flask and allowed to grow till confluency
b) Once the cells reached confluency, trypsinized and seeded in 6-well cell culture plates. The cells were allowed to grow till 95-100% confluency (but NOT over-confluent)
c) Media was discarded from the wells and washed with PBS. Next, titrated Influenza
virus in growth media (DMEM+1% BSA+P/S+ 1µg/ml TPCK treated trypsin)
d) Once the cells were ready, the paper-strip at different sizes and the virus were co incubated.
Effective concentration of test compound present in each strip:
1 strip=0.2 cm X0 .2 cm=0.04cm2 or 4 mm2
4mm2 contain 0.016 mg or 16µg compound
i.e. 1strip contain 16 µg compound
2 strips contain 32 µg compound
6 strips contain 96 µg compound
For test compound strips were (1, 2 or 3 in nos) incubated with 100 µL of viral growth media containing 50TICD50 of virus, therefore the effective concentration of test compound was 16/32/48µg for 100µL.
IC50 of the compound was 490µg/mL i.e. 49µg/100µL and we used the test compound =IC50.
Briefly, coated papers were cut in four (4) 1-inch strips in sterile condition and kept on sterile dish. Virus was added (same volume and TICD50 dose) onto the paper strips dropwise to cover the strip completely. After 5 minutes the coated materials released into the media containing virus was next added to each well. No compound but virus and only virus were kept as controls.
e) Allowed to incubate for 1.5/2 hr at 37 0C with 5 % CO2 in a CO2 incubator.
f) Following the media was discarded and fresh influenza growth media (with TPCK treated trypsin) was added and incubated for 48 hrs.
g) After 48 hrs, standard MTS assay was performed to check cell viability.
h) For MTS assay:
i) Discard old media
i. Wash cells with PBS (tap gently on tissue to remove dead cells)
ii. Prepare fresh media ((DMEM+10% FBS+P/S) with 1 mg/ml MTT dye
iii. Add to the cells and incubate for 3 hrs
iv. After 3 hrs discard media
v. Add DMSO and read absorbance at 595nm
3. Detection of viral nuclear protein by immuno-fluorescence microscopy
a) An indirect immunofluorescence assay was performed using H1N1 specific anti-NP antibody to detect viral NP protein in cells treated with test compound plus virus, virus only and test compound only
b) Briefly, MDCK cells were grown on coverslips in 24-well well plates for 24 h followed by specific treatment
c) After washing cells were fixed with 4 % paraformaldehyde (PFA) for 15 min at RT, permeabilized with ice-cold acetone for 30 min and incubated in blocking solution (1 % BSA in PBS-T) at 37 °C for 1 h. Rabbit polyclonal anti-NP antibody (1:1000, Sino Biologicals, Japan) were then added to the cells (4 °C overnight).
d) The next day, the cells were incubated with FITC-labelled anti-Rabbit IgG (Fab2, 1:1000; Thermo Fisher Scientific) at RT for 1 h in the dark.
e) Following, they were washed with PBS-T and counter-stained with 4’,6-diamidino-2-
phenylindole-dihydrochloride (DAPI) to detect nuclei and mounted on glass slide using VectaShield mounting media (Vector Laboratories, USA).

Results
Fig.3 illustrates the Cytotoxicity of the drug in MDCK cells as determined by MTT assay. Cells were treated with the drug ranging from 20 µg/ml to 1200 µg/ml for 24 h. Next, the cells were washed with PBS and incubated with 10 µL of MTT solution (1 mg/ml) for 3 h. Following incubation cells were dissolved in DMSO and the absorbance was read at 595 nm. IC50 value of drug was determined to be 490 µg/ml for MDCK. Each data point represents the mean of cell viability ± SD relative to the untreated control (cells with media only) normalized to 100% of experiment in quadruplicate.
In vitro protection of MDCK cells against H1N1 influenza virus: Data represented in figure 4 is expressed as percentage (%) of MDCK cell protection. Prior adding to cells influenza A/PR/8/34 (H1N1) viral (at 50TCID50 /ml) was incubated with test compound (16, 32, and 48µg/100µl) for 5 min in complete DMEM media. Following the treated virus was added to the cells and incubated for 1-2 hours. Protective efficacy was evaluated by performing standard MTT assay. Each bar represents mean protection %±SE of five independent experiments (n=28) performed under analogous conditions. Data indicate that higher level of protection in a dose dependent manner. The percent protection (% protection) was calculated as [(A–B)/CB)] x100, where A, B and C represent the absorbance of treated infected, untreated infected and untreated uninfected cells, respectively.
Fig. 5 illustrates the indirect immunofluorescence assay showing the antiviral efficacy of test compound in MDCK cells against H1N1 virus: Immunofluorescence assay (IFA) using an anti-NP antibody. Images were captured in ZEISS LSM 980 Confocal Laser Scanning Microscope (60x magnification, FITC and DAPI filter). Minor signals in cells infected with virus pre-treated with test compound (48µg/100µL,