Polymeric particulate carrier

A polymeric carrier is disclosed having a particle size such that the largest dimension thereof is less than about 8 mm and the smallest dimension is at least about 0.1 mm. The carrier comprises a plurality of polymeric segments containing chemically modifiable groups on the surface. The segments are united into a coherent, stable porous matrix by means of cross-links between segments. The matrix defines at least one cavity of predetermined size, and the particle size of the carrier, the porosity of the matrix and the presence of the cavity combine to provide desirable liquid compatibility and diffusional characteristics.

The present invention relates to polymeric carriers containing chemically 
modifiable groups and, more particularly, to porous carriers which provide 
desirable fluid compatability and diffusional characteristics. 
Carriers, sometimes referred to as solid resins or supports, are currently 
being used in a number of applications including the solid phase synthesis 
of organic compounds such as polypeptides, enzyme immobilization, 
chromatography, ion-exchange, and solid phasesequencing of proteins, 
peptides and nucleic acids. The carriers are generally polymeric or 
inorganic and contain covalently attached chemically modifiable groups, 
the nature of the groups depending upon the particular application for 
which the carrier is intended. In application, the modifiable groups may 
undergo covalent or ionic modification. Advantages accompanying the use of 
carriers are, among others, ease in separation and purification of 
synthetic intermediates, minimization of the likelihood that valuable 
products may be lost, and enhanced stability and durability of catalytic 
substances attached to the carrier. 
Heretofore, limitations accompanying the use of carriers have centered on 
their liquid compatibility and diffusional characteristics. Ideally, a 
carrier should be compatible with both aqueous and organic liquids and, 
particularly, polar aqueous or organic solvents. As to diffusion, liquid 
should be able to freely permeate the carrier so as to provide access to 
the modifiable groups at the interior. 
The problem has resided in obtaining a carrier wherein both desirable 
liquid compatibility and diffusional characteristics are present in 
combination with good mechanical and chemical stability. For example, the 
customary carrier for solid-phase peptide synthesis is a chemically 
modified polystyrene resin. To provide desirable diffusional 
characteristics, this material must be swollen with a non-polar organic 
solvent. In turn, this has limited the solid-phase synthesis of 
polypeptides to those techniques which are accomplished in an organic 
environment. Another customary carrier is derivatized porous glass. While 
this carrier is compatible with either water or organic solvents, optimum 
stability characteristics have been difficult to achieve. 
Accordingly, the principal objective of the present invention is to provide 
a carrier which is useful in applications such as heretofore identified, 
and which possesses both desirable liquid compatibility and diffusional 
characteristics in combination with good mechanical and chemical 
stability. Other objectives and advantages of the present invention will 
become apparent upon reading the following description of the invention 
taken in combination with the attached drawing wherein FIGS. 1 and 3 
schematically illustrate embodiments of carriers of the invention, FIG. 1 
is a sectional view taken along line 2--2 of FIG. 1. FIG. 4 is a partial 
sectional view taken along line 4--4 of FIG. 3, and FIG. 5 is a sectional 
view taken along line 5--5 of FIG. 3. 
In accordance with the present invention, a carrier of predetermined 
particle size is provided which is formed from a plurality of polymeric 
segments having chemically modifiable groups on their surface. The 
segments are united into a coherent, stable porous matrix by means of 
cross-links between them. The matrix defines at least one cavity of 
predetermined size. The particle size of the carrier, the porosity of the 
matrix and the presence of the cavities combine to provide desirable 
liquid compatibility and diffusional characteristics. 
Being of known size, porosity, and density, the carriers can be combined to 
form the particulate assemblies having tailored diffusional 
characteristics. Such assemblies are comprised of a plurality of the 
carriers as individual particles, and can either be a homogeneous 
population of substantially identical particles or a mixture of particles 
having different, but known, size, porosity and density. 
Turning to the drawing, FIG. 1 schematically illustrates a carrier 8 
generally in the shape of a hollow spherical particle, the surface of 
which contains a plurality of polymeric segments 10 which contain 
chemically modifiable groups x exposed on the surface thereof. In order to 
unite the polymeric segments 10 into a coherent matrix to provide a 
self-supporting stable particulate structure with desirable liquid 
compatibility features, the segments 10 are joined together by means of 
chemical cross-links 12 between the segments. As illustrated, the matrix 
of segments and cross-links contains open spaces 14 such that, although 
the particulate carrier is mechanically and chemically stable, the carrier 
is nevertheless porous to liquids. Referring specifically to FIG. 2, the 
matrix formed by the polymer segments 10 and the cross-links 12 defines a 
cavity 16. As will be discussed hereafter, the size of the cavity 16 can 
be predetermined and, accordingly, the carrier can be fashioned with 
desirable fluid diffusional characteristics. 
Turning now to FIGS. 3-5, a further schematic illustration of a particulate 
carrier constructed in accordance with this invention is illustrated. In 
this embodiment, three additional cavities, 18, 20, and 22, are shown. In 
turn, each of these cavities is defined by the coherent, porous matrix 
formed by the polymer segments 10 and cross-links 12 between the segments. 
As with FIGS. 1 and 2, the segments contain chemically modifiable groups x 
exposed on the surface of the segments. 
A useful method for forming polymeric particulate carriers of the instant 
invention involves contacting a polymer, dissolved in an appropriate 
solvent, with a dissolvable, generally inorganic, particulate solid 
support such that the polymer is adsorbed to the surface of the support 
particles. After removing any excess polymer which is not adsorbed, the 
polymer is cross-linked and, thereafter, the supporting member is 
dissolved to leave the carrier. Gentle agitation while the support is 
suspended in a fluid should be employed during the cross-linking reaction 
so that cross-linking only occurs between polymer segments adsorbed on 
individual support particles, and not between polymer segments adsorbed on 
different particles. 
The size, shape, and porosity of the support is selected in accordance with 
the desired size, shape and cavity configuration of the carrier. It can be 
solid, as when a carrier with a single cavity such as illustrated in FIG. 
1 is desired, or porous where two or more cavities are to be present (FIG. 
3). 
An important aspect of this invention resides in the fact that the carrier 
is particulate and that its particle size and the size of the cavity(s) 
contained therein are predeterminable; these factors, in combination with 
the degree of cross-linking achieved, influencing the carrier's liquid 
diffusional characteristics. In accordance with this invention, particle 
size will be such that the largest dimension of the carrier will be less 
than about 8 mm and, preferably, less than about 3.0 mm. In general, the 
smallest dimension will be at least about 0.1 mm. 
Where the carrier contains only a single cavity, i.e., formed from a solid 
support particle, the size of that cavity will closely approximate the 
size of the dissolvable support particle since the matrix itself is 
generally very thin, e.g., less than about 200 A thick. However, where 
more than one cavity is present, cavity size cannot be directly related to 
the particle size of the support. And, in this latter instance, cavity 
size is best defined by the smallest dimension of the cavities which, for 
the present invention, is at least about 50 A and, preferably, at least 
about 300 A. In general, the ratio of carrier polymer to void space is 
less than about 10% by volume and, preferably, less than about 5%. 
Turning to the polymers useful in forming carriers of the invention, the 
important aspects thereof are that the polymer can be adsorbed to a 
support, that, after adsorption, it can be cross-linked into a porous 
matrix, and that it contains or can be fashioned to contain, the desired 
chemically modifiable surface group. For most applications, the polymer 
will, prior to adsorption, contain both the functional groups needed to 
achieve cross-linking and the groups which are intended for chemical 
modification in the end use application. 
For example, in the event that the carrier is to be used in applications 
such as peptide synthesis or sequencing or enzyme immobilization, wherein 
the chemically modifiable group is an amine, then polymers having free 
reactive amine groups are most useful for the carrier. By selecting an 
appropriate cross-linking reagent, such as a dialdehyde, formaldehyde, 
phosgene, thiophosgene, etc., a portion of the amine groups on the polymer 
can be involved in the cross-linking reaction leaving other of the amine 
groups for the desired attachment to the carboxyl group of an amino acid, 
peptide fragment or enzyme. A particularly useful polymer is 
polyethyleneimine because of the large number of reactive primary amine 
groups in the polymer. 
Proteins themselves are useful polymers where chemically modifiable amine 
groups are desired, the protein again being conveniently cross-linked on a 
support with a dialdehyde. Human serum albumin (HSA) is considered to be 
an especially useful protein for this application. As a particulate 
carrier, this protein which is biodegradable may find use as an 
encapsulating device for the sustained in-vivo release of biologicals such 
as drugs, hormones, and the like. 
Polyamide (partially hydrolyzed) is a further type of useful polymer. This 
polymer contains both free acid groups and free amine groups. Thus, 
depending on the cross-linking agent utilized, the carrier can contain, as 
the chemically modifiable group, either an acid or amine. If an amine is 
desired, then a diamine can be used as the cross-linking agent. If, on the 
other hand, chemically modifiable acid groups are desired, then a 
dialdehyde, formaldehyde, etc. can be used for cross-linking. 
An advantage accompanying the use of a polymer such as a polyamide having 
dual functionality is that the availability of the desired chemically 
modifiable group is not effected by the cross-linking operation, and as a 
result, large excesses of the cross-linking reagent can be employed. This 
is not the case with respect to the use of polymers wherein cross-linking 
is through the same type of group which is also to be available for 
chemical modification during use of the carrier. In this latter instance, 
the degree of cross-linking effected must be controlled so as to have a 
sufficient number of chemically modifiable groups left in the carrier so 
that it is useful for its intended application. 
As will be appreciated, a number of other polymers are useful in forming 
the carriers of the instant application. Where chemically modifiable OH 
groups are desired, polyvinyl alcohol or dextran can be used, and 
cross-linking accomplished with, for example, a dihalide or diisocyanate. 
Where free acid groups are required, in addition to the above mentioned 
polyamide polymer, polyacrylic acid and polyethylene-maleic anhydride are 
also useful. In these latter instances, a diamine can be used for 
cross-linking. 
While the foregoing discussion has centered on the use of polymers which, 
as prepared, contain the chemically modifiable group, the invention also 
embraces polymeric particulate carriers wherein the modifiable group is 
introduced after polymer formation. Carriers containing groups chemically 
modifiable by ionization and useful in ion exchange chromatography are 
examples. Thus, if the carrier contains free amine groups, it can be 
directly used as a basic ion exchange resin. However, by reacting the 
modifiable amine groups with, for example, succinic anhydride, a carrier 
useful as an acidic ion exchange resin is provided. 
Turning to the dissolvable support, particularly useful materials are 
alumina, silica and glass. These materials are commercially available or 
can be easily prepared, in a variety of shapes and with controlled pore 
size so as to make the volume of the cavities in the polymeric carrier 
easily determinable. With these materials, removal from the cross-linked 
polymer can be accomplished by dissolution using solvents which do not 
adversely affect the cross-linked polymer. Glass and silica can be 
conveniently dissolved with a strong base such as sodium hydroxide while 
alumina can be dissolved with a strong acid such as HCl.

The following examples illustrate the present invention. All parts and 
percentages are by weight unless otherwise indicated. Temperature is in 
degrees centigrade. 
EXAMPLE I 
400 ml of an 8.3% solution of polyethyleneimine (PEI-600 Dow) in methanol 
was mixed with 100 ml of porous approximately spherical alumina beads 
(350-500 A pore size, about 1 mm diameter particle size). Trapped air was 
removed by vacuum and the mixture gently agitated for 30 minutes. The 
beads were washed with 5 portions of methanol (200 ml each) and then dried 
in vacuo at room temperature. The beads (25 g, containing adsorbed PEI), 
were reacted at room temperature with 250 ml of 0.4% aqueous 
glutaraldehyde to cross-link the polymer. Gentle agitation accompanied the 
cross-linking reaction. Trapped air was removed and the reaction was 
continued for 30 min. The glutaraldehyde solution was then replaced with 
100 ml of an 8.3% solution of PEI in methanol to react with any 
glutaraldehyde which failed to completely react, and after 15 min. of 
gentle agitation, the PEI solution was replaced with 100 ml of methanol. A 
total of 2 g of NaBH.sub.4 was added in small increments over a period of 
30 min. to form stable cross-links. The beads were then washed and dried 
as above described. A PEI carrier was then produced by treatment of 10 
grams of the beads with 100 ml of 1N HCl for approximately 15 min. at room 
temperature. The acid was decanted and replaced with a second 100 
ml-portion of 1N HCl. After 30 min. the PEI-carrier containing amine 
modifiable groups was then washed with water and methanol and then dried 
in vacuo at room temperature. 
To demonstrate the use of this carrier in connection with enzyme-like 
catalysis, histidyl residues were introduced onto the carrier by reaction 
of the amine groups with the nitrophenyl ester, Boc-His (.sup.Im 
DNP)-ON.sub.p, as follows: After treatment with 0.2 ethanolic-KOH, the PEI 
carrier (4 ml settled volume) was mixed with 100 mg of the nitrophenyl 
ester and 2 equivalents of triethylamine in 5 ml of dry dioxane, and the 
mixture was rotated for 12 hr at 60.degree.. The boc group was removed 
with 30% TFA in CHCl.sub.3 (5 ml, 12 hr, at 25.degree.). The DNP group was 
removed bythiolysis (0.1 M mercaptoethenol, pH 9, 12 hr, 25.degree.). 
After hydrolysis (6N HCl, 110.degree., 24 hr), amino acid analysis was 
done. The result was 0.4 meg His/g. Lauroyl groups were then introduced by 
treatment with nitrophenyllaurate (500 mg in 25 ml of dioxane, containing 
0.2 ml of triethylamine) for 72 hours at 70.degree.. A sample which 
contained no histidyl residues was lauroylated in the same way and used as 
a control. 
p-Nitrotrifluoroacetanilide was hydrolyzed using the following conditions: 
0.01 M N-ethylmorpholine-HCl buffer, pH 8.1, 40 ml of substrate, 10.sup.-4 
M; 25.degree.; 7 mg of catalyst. At timed intervals samples were removed 
for analysis by means of a syringe fitted with a tube covered by nylon 
net. The second-order rate constants for hydrolysis catalyzed by 
imidazole, and the lauroyl-histidyl-PEI-carrier were estimated to be 0.007 
M.sup.-1 s.sup.-1 and 1.7 M.sup.-1 s.sup.-1, respectively. The rate 
constants were calculated on the basis of total imidazole content in both 
cases. Carriers modified only with lauroyl groups do not produce a rate 
enhancement over background. The sizable rate enhancement about 230 times 
for the lauroyl, histidyl modified carrier is believed to be attributable 
to the lauroyl groups being able to bind substrate in close proximity to 
imidazole ring of histidine which, in turn, is obtainable because of the 
desirable fluid diffusion characteristics of the carrier. 
The enzyme, pepsin, was also immobilized on the PEI-carrier of Example I by 
washing 400 mg of the carrier with a coupling buffer (0.13 N pyridine-HCl, 
pH 4.5) and then adding the carrier to a solution (3.5 ml) of pepsin (2 
mg/ml) in the coupling buffer. The suspension so formed was then combined 
with 14 mg of the water soluble carbodiimide, 1 
-cyclohexyl-3-(2-morpholinoethyl) carbodiimide-metho-p-toluene sulphonate. 
This suspension was maintained at pH 4.5 for 1.5 hrs at room temperature 
and then rotated overnight at 0-4.degree.. After successive washing with 
0.5 l portions of 1 mM HCl, 0.5M KCl and 1 mM HCl, thePEI-immobilized 
pepsin exhibited high activity. 
EXAMPLE II 
Controlled-pore glass (10 g, 550 A, 40/80 mesh) and 100 ml of 
polyethyleneimine (50,000 MW, 8% in methanol) were placed in a round 
bottom flask. A vacuum was applied for 10 min with rotation of the flask. 
After an additional hour of rotation, the solution was decanted. The beads 
were washed twice with methanol (at least 100 ml each time) and dried in 
vacuo for 24 hr. The polymer layer was cross-linked with glutaraldehyde 
using the following conditions: 80 ml of N-ethyl morpholine (0.1M, pH 8.0) 
containing 0.2% glutaraldehyde, 5 min evacuation, rotationovernight, and 
washing with buffer. The inorganic core was removed by treatment with 250 
ml of 2 N NaOH for 24 hrs to provide the carrier. 
EXAMPLE III 
Polyvinylimidazole (PVI) was absorbed to porous glass beads as described in 
Example II, and cross-linking accomplished with diazotized 4, 4' sulfonyl 
dianiline. To 5 g of PVI-Glass suspended in 20 ml of 0.1 phosphate buffer 
(pH 0.8), 70.mu. moles of diazonium salt was added. The mixture was 
tumbled gently at 0-4.degree. for 90 min. The beads were washed with 3, 
200-ml portions each of the following: phosphate buffer, H.sub.2 O and 
methanol. The material was dried in vacuo overnight. Removal of the 
inorganic core was accomplished with base as described in Example II. 
EXAMPLE IV 
A solution of HSA (about 1% in water, 50 ml) was suspended with 100 ml of 
alumina beads as described in Example I; the beads having first been 
washed with distilled water and trapped air removed by vacuum. After about 
30 minutes of gentle agitation, the procedure described in Example I was 
continued except that about a 1% aqueous solution of HSA was used to "cap" 
any glutaraldehyde which failed to react. Removal of the alumina support 
and recovery of the cross-linked HSA carrier was as described in Example 
I. 
While the present invention has been described in connection with certain 
preferred embodiments, it is to be understood that it is not to be limited 
to only those embodiments. On the contrary, it is intended to cover all 
modifications and alternatives falling within the spirit and scope of the 
invention as expressed in the appended claims.